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.

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.

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.

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.

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.

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.

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.

Design and synthesis of trivalent ligands targeting opioid, cholecystokinin, and melanocortin receptors for the treatment of pain.

It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine's analgesic efficacy by reducing serious side effects such as tolerance and addiction. Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors.

Synthesis and evaluation of new endomorphin analogues modified at the Pro(2) residue.

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Delta(3)Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (DeltaAla) have been used to prepare [Delta(3)Pro(2)]EM-2 (1), [Aze(2)]EM-1 (2), [Aze(2)]EM-2 (3), [3Aze(2)]EM-1 (4), [3Aze(2)]EM-2 (5) and [DeltaAla(2)]EM-2 (6). Binding assays and functional bioactivities for mu- and delta-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue.

The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety.

In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[D-Cys-Gly-Phe-Met-Pro-D-Cys]-Trp-NH-[3',5'-(CF(3))(2)Bzl]) was found as a highly selective delta opioid agonist over mu receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24h half life in rat plasma.

Synthesis and investigations of double-pharmacophore ligands for treatment of chronic and neuropathic pain.

Acids 9a-f as possible bivalent ligands designed as a structural combination of opioid mu-agonist (Fentanyl) and NSAID (Indomethacin) activities and produced compounds which were tested as analgesics. The obtained series of compounds exhibits low affinity and activity both at opioid receptors and as cyclooxygenase (COX) inhibitors. One explanation of the weak opioid activity could be stereochemical peculiarities of these bivalent compounds which differ significantly from the fentanyl skeleton. The absence of significant COX inhibitory properties could be explained by the required substitution of an acyl fragment in the indomethacin structure for 4-piperidyl.

A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists.

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH(2)) showed potent binding and agonist activities at delta and mu opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands.

Role of benzimidazole (Bid) in the delta-opioid agonist pseudopeptide H-Dmt-Tic-NH-CH(2)-Bid (UFP-502).

H-Dmt-Tic-NH-CH(2)-Bid (UFP-502) was the first delta-opioid agonist prepared from the Dmt-Tic pharmacophore. It showed interesting pharmacological properties, such as stimulation of mRNA BDNF expression and antidepression. To evaluate the importance of 1H-benzimidazol-2-yl (Bid) in the induction of delta-agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S transforms the reference delta-agonist into delta-antagonists. Phenyl ring of benzimidazole is not important for delta-agonism; in fact 1H-imidazole-2-yl retains delta-agonist activity.

Development of novel enkephalin analogues that have enhanced opioid activities at both mu and delta opioid receptors.

Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve therapeutic benefit for the treatment of pain due to mixed mu and delta opioid agonist activities. Ligand 16, in which a Dmt-substituted enkephalin-like structure was linked to the N-phenyl-N-piperidin-4-yl propionamide moiety, showed very high binding affinities (0.4 nM) at mu and delta receptors with an increased hydrophobicity (aLogP = 2.96). This novel lead compound was found to have very potent agonist activities in MVD (1.8 nM) and GPI (8.5 nM) assays.

Synthesis of stable and potent delta/mu opioid peptides: analogues of H-Tyr-c[D-Cys-Gly-Phe-D-Cys]-OH by ring-closing metathesis.

Ring-closing metathesis has emerged as a powerful tool in organic synthesis for generating cyclic structures via C-C double bond formation. Recently, it has been successfully used in peptide chemistry for obtaining cyclic molecules bridged through an olefin unit in place of the usual disulfide bond. Here, we describe this approach for obtaining cyclic olefin bridged analogues of H-Tyr-c[D-Cys-Gly-Phe-Cys]-OH. The synthesis of the new ligands was performed using the second generation Grubbs' catalyst. The resulting cis-8 (cDADAE) and trans-9 (tDADAE) were fully characterized and tested at delta, mu, and kappa opioid receptors. Also the linear precursor 13 (lDADAE) and the hydrogenated derivative 11 (rDADAE) also were tested. All the cyclic products containing a olefinic bond are slightly selective but highly active and potent for the delta and mu opioid receptors. Activity toward the kappa opioid receptors was absent or very low.

Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for delta/mu opioid receptor agonists and neurokinin-1 receptor antagonists.

A series of bifunctional peptides that act as agonists for delta and mu opioid receptors with delta selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at delta/mu opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 (7) had excellent agonist activity for both delta opioid and mu opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states.