Potent Dmt-Tic pharmacophoric delta- and mu-opioid receptor antagonists

A series of dimeric Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues (8-14, 18-22) were covalently linked through diaminoalkane and symmetric or asymmetric 3,6-diaminoalkyl-2(1H)-pyrazinone moieties. All the compounds exhibited high affinity for both delta-opioid receptors [Ki(delta) = 0.06-1.53 nM] and mu-opioid receptors [Ki(mu) = 1.37-5.72 nM], resulting in moderate delta-receptor selectivity [Ki(mu)/Ki(delta) = 3-46]. Regardless of the type of linker between the Dmt-Tic pharmacophores, delta-opioid-mediated antagonism was extraordinarily high in all analogues (pA2 = 10.42-11.28), while in vitro agonism (MVD and GPI bioassays) was essentially absent (ca. 3 to >10 microM). While an unmodified N-terminus (9, 13, 18) revealed weak mu-opioid antagonism (pA2 = 6.78-6.99), N,N'-dimethylation (21, 22), which negatively impacts on mu-opioid-associated agonism (Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435-5441), markedly enhanced mu-opioid antagonism (pA2 = 8.34 and 7.71 for 21 and 22, respectively) without affecting delta-opioid activity. These data are the first evidence that a single dimeric opioid ligand containing the Dmt-Tic pharmacophore exhibits highly potent delta- and mu-opioid antagonist activities.     

Development of potent mu-opioid receptor ligands using unique tyrosine analogues of endomorphin-2

Six analogues of tyrosine, which contained alkyl groups at positions 2', 3', and 6', either singly or in combination on the tyramine ring, were investigated for their effect on the opioid activity of [Xaa(1)]endomorphin-2 (EM-2). The opioid analogues displayed the following characteristics: (i) high mu-opioid receptor affinity [K(i)(mu) = 0.063-2.29 nM] with selectivity [K(i)(delta)/K(i)(mu)] ranging from 46 to 5347; (ii) potent functional mu-opioid agonism [GPI assay (IC(50) = 0.623-0.924 nM)] and with a correlation between delta-opioid receptor affinities and functional bioactivity using MVD; (iii) intracerebroventricular administration of [Dmt(1)]- (14) and [Det(1)]EM-2 (10) produced a dose-response antinociception in mice, with the former analogue more active than the latter; and (iv) a marked shift occurred from the trans-orientation at the Tyr(1)-Pro(2) bond to a cis-conformer compared to that observed previously with [Dmt(1)]EM-2 (14) (Okada et al. Bioorg. Med. Chem. 2003, 11, 1983-1984) except [Mmt(1)]EM-2 (7). The active profile of the [Xaa(1)]EM-2 analogues indicated that significant modifications on the tyramine ring are possible while high biological activity is maintained.     

Further studies on the Dmt-Tic pharmacophore: hydrophobic substituents at the C-terminus endow delta antagonists to manifest mu agonism or mu antagonism

Twenty N- and/or C-modified Dmt-Tic analogues yielded similar K(i) values with either [(3)H]DPDPE (delta(1) agonist) or [(3)H]N, N(Me)(2)-Dmt-Tic-OH (delta antagonist). N-Methylation enhanced delta antagonism while N-piperidine-1-yl, N-pyrrolidine-1-yl, and N-pyrrole-1-yl were detrimental. Dmt-Tic-X (X = -NHNH(2), -NHCH(3), -NH-1-adamantyl, -NH-tBu, -NH-5-tetrazolyl) had high delta affinities (K(i) = 0.16 to 1 nM) with variable mu affinities to yield nonselective or weakly mu-selective analogues. N, N-(Me)(2)Dmt-Tic-NH-1-adamantane exhibited dual delta and mu receptor affinities (K(i)delta = 0.16 nM and K(i)mu = 1.12 nM) and potent delta antagonism (pA(2) = 9.06) with mu agonism (IC(50) = 16 nM). H-Dmt-betaHTic-OH (methylene bridge between C(alpha) of Tic and carboxylate function) yielded a biostable peptide with high delta affinity (K(i) = 0.85 nM) and delta antagonism (pA(2) = 8.85) without mu bioactivity. Dmt-Tic-Ala-X (X = -NHCH(3), -OCH(3), -NH-1-adamantyl, -NHtBu) exhibited high delta affinities (K(i) = 0.06 to 0.2 nM) and elevated mu affinities (K(i) = 2.5 to 11 nM), but only H-Dmt-Tic-Ala-NH-1-adamantane and H-Dmt-Tic-Ala-NHtBu yielded delta receptor antagonism (pA(2) = 9.29 and 9.16, respectively). Thus, Dmt-Tic with hydrophobic C-terminal substituents enhanced mu affinity to provide delta antagonists with dual receptor affinities and bifunctional activity.     

Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore

Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.     

Potent delta-opioid receptor agonists containing the Dmt-Tic pharmacophore

Conversion of delta-opioid receptor antagonists containing the 2',6'-dimethyl-L-tyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore into potent delta-agonists required a third heteroaromatic nucleus, such as 1H-benzimidazole-2-yl (Bid) and a linker of specified length both located C-terminally to Tic in the general formula H-Dmt-Tic-NH-CH(R)-R'. The distance between Tic and Bid is a determining factor responsible for the acquisition of delta agonism (2, 2', 3, 4, 6) or delta antagonism (8). Compounds containing a C-terminal Ala (1, 1'), Asp (5), or Asn (7) with an amide (1, 1', 5) or free acid group (7) served as delta-antagonist controls lacking the third heteroaromatic ring. A change in chirality of the spacer (2, 2') or inclusion of a negative charge via derivatives of Asp (4, 6) resulted in potent delta agonism and moderate mu agonism, although delta-receptor affinity decreased about 10-fold for 4 while mu affinity fell by over 2 orders of magnitude. Repositioning of the negative charge in the linker altered activity: H-Dmt-Tic-NH-CH(CH(2)-Bid)COOH (6) maintained high delta affinity (K(i) = 0.042 nM) and delta agonism (IC(50) = 0.015 nM), but attachment of the free acid group to Bid [H-Dmt-Tic-NH-CH(2)-Bid(CH(2)-COOH) (9)] reconstituted delta antagonism (K(e) = 0.27 nM). The data demonstrate that a linker separating the Dmt-Tic pharmacophore and Bid, regardless of the presence of a negative charge, is important in the acquisition of opioids exhibiting potent delta agonism and weak mu agonism from a parent delta antagonist.     

Bifunctional [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed mu-agonist/delta-antagonist and dual mu-agonist/delta-agonist opioid ligands

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, containing alkylated Phe3 derivatives, 2'-monomethyl (2, 2'), 3',5'- and 2',6'-dimethyl (3, 3', and 4', respectively), 2',4',6'-trimethyl (6, 6'), 2'-ethyl-6'-methyl (7, 7'), and 2'-isopropyl-6'-methyl (8, 8') groups or Dmt (5, 5'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).     

Development of potent bifunctional endomorphin-2 analogues with mixed mu-/delta-opioid agonist and delta-opioid antagonist properties

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited mu-opioid receptor affinity in the nanomolar range (K(i) = 2.41-6.59 nM), which, however, was 3- to 10-fold less than the parent peptide. Replacement of Tyr(1) by Dmt (2',6'-dimethyl-l-tyrosine) (19-32) exerted profound effects: (i) acquisition of high mu-opioid receptor affinity (K(i) = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional mu-opioid receptor agonism (IC(50) < 1 nM) for 19 ([Dmt(1)]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak delta-opioid antagonist activity (pA(2) = 5.41-7.18) except 19 ([Dmt(1)]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent delta-agonism (IC(50) = 0.62 nM, pA(2) = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr(1) analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt(1)]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent mu-opioid receptor bioactivity and dual functional agonism.     

Synthesis and pharmacological evaluation of novel octahydro-1H-pyrido[1,2-a]pyrazine as mu-opioid receptor antagonists

To better understand structural requirements for a mu ligand of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class to interact with the mu opioid receptor, we have described in the previous article (Le Bourdonnec, B. et al. J. Med. Chem. 2006, 25, 7278-7289) new, constrained analogues of the N-phenethyl derivative 3. One of the active constrained analogues, compound 4, exhibited subnanomolar mu-opioid receptor affinity (K(i) = 0.62 nM) and potent mu-opioid antagonist activity (IC(50) = 0.54 nM). On the basis of structure 4, a new series of mu-opioid receptor antagonists were designed. In these compounds the octahydroquinolizine template of 4 was replaced by an octahydro-1H-pyrido[1,2-a]pyrazine scaffold. The new derivatives were tested for their binding affinities and in vitro functional activity against the cloned human mu-, delta-, and kappa-opioid receptors. From this study, we identified compound 36, which displays high affinity toward the mu-opioid receptor (K(i) = 0.47 nM), potent mu in vitro antagonist activity (IC(50) = 1.8 nM) and improved binding selectivity profile mu/kappa and mu/delta, when compared to 4.     

Crystal structures of dipeptides containing the Dmt-Tic pharmacophore

The crystal structures of three analogues of the potent delta-opioid receptor antagonist H-Dmt-Tic-OH (2',6'-dimethyl-L-tyrosine-L-1,2,3,4-tetrahydroisoquinoline-3-carboxylate), N,N (CH(3))(2)-Dmt-Tic-OH (1), H-Dmt-Tic-NH-1-adamantane (2), and N,N(CH(3))(2)-Dmt-Tic-NH-1-adamantane (3) were determined by X-ray single-crystal analysis. Crystals of 1 were grown by slow evaporation, while those of 2 and 3 were grown by vapor diffusion. Compounds 1 and 3 crystallized in the monoclinic space group P2(1), and 2 crystallized in the tetragonal space group P4(3). Common backbone atom superimpositions of structures derived from X-ray diffraction studies resulted in root-mean-square (rms) deviations of 0.2-0.5 A, while all-atom superimpositions gave higher rms deviations from 0.8 to 1.2 A. Intramolecular distances between the aromatic ring centers of Dmt and Tic were 5.1 A in 1, 6.3 A in 2, and 6.5 A in 3. The orientation of the C-terminal substituent 1-adamantane in 2 and 3 was affected by differences in the psi torsion angles and strong hydrogen bonds with adjacent molecules. Despite the high delta-opioid receptor affinity exhibited by each analogue (K(i) < 0.3 nM), high mu receptor affinity (K(i) < 1 nM) was manifested only with the bulky C-terminal 1-adamantane analogues 2 and 3. Furthermore, the bioactivity of both 2 and 3 exhibited mu-agonism, while 3 also had potent delta-antagonist activity. Those data demonstrated that a C-terminal hydrophobic group was an important determinant for eliciting mu-agonism, whereas N-methylation maintained delta-antagonism. Furthermore, the structural results support the hypothesis that expanded dimensions between aromatic nuclei is important for acquiring mu-agonism.     

Extended TIP(P) analogues as precursors for labeled delta-opioid receptor ligands

Tyr-Tic-Phe-Phe-OH (TIPP) and the shorter Tyr-Tic-Phe-OH (TIP) peptides are potent and highly selective antagonists at the delta-opioid receptor and, therefore, are ideal candidates for the attachment of labels to assist in the study of delta-opioid receptors. Peptides extended at the C-terminus with residues which can be used as handles for further modification and/or labeling (i.e. Asx, Glx, and Lys) were synthesized. The TIPP-D/L-Asx/Glx derivatives exhibited similar delta-receptor affinity to TIPP (K(i) = 5-10 nM vs K(i) = 6 nM), and neither the location of the carboxylic acid moiety nor the stereochemistry of the C-terminal residue significantly affected the delta-receptor affinity of these derivatives. Extension of TIPP with an additional residue did not increase mu-receptor affinity, even though the position of the acidic group, which imparts delta-receptor selectivity to TIPP, was shifted relative to the carboxylic acid moiety of TIPP. The delta-receptor affinities of the TIP-D/L-Asx/Glx derivatives were found to be influenced mainly by the position of the carboxylic acid function rather than the stereochemistry of the C-terminal residue. TIP(P)-D/L-Lys(Ac)-OH derivatives exhibited moderate delta-receptor affinity (K(i)(delta) = 16-28 nM). The most potent compounds found in the extended TIP(P) series were TIPP-D-Gln-OH and TIP-D-Gln-OH (K(i)(delta) = 5 nM) which had similar affinities to TIPP.     

Evaluation of the Dmt-Tic pharmacophore: conversion of a potent delta-opioid receptor antagonist into a potent delta agonist and ligands with mixed properties.

Analogues of the 2',6'-dimethyl-L-tyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore were prepared to test the hypothesis that a "spacer" and a third aromatic center in opioid peptides are required to convert a delta-antagonist into ligands with delta-agonist or with mixed delta-antagonist/mu-agonist properties. Potent delta-agonists and bifunctional compounds with high delta- and mu-opioid receptor affinities were obtained by varying the spacer length [none, NH-CH(2), NH-CH(2)-CH(2), Gly-NH-CH(2)] and C-terminal aromatic nucleus [1H-benzimidazole-2-yl, phenyl (Ph) and benzyl groups]. C-terminal modification primarily affected mu-opioid receptor affinities, which increased maximally 1700-fold relative to the prototype delta-antagonist H-Dmt-Tic-NH(2) and differentially modified bioactivity. In the absence of a spacer (1), the analogue exhibited dual delta-agonism (pEC(50), 7.28) and delta-antagonism (pA(2), 7.90). H-Dmt-Tic-NH-CH(2)-1H-benzimidazole-2-yl (Bid) (2) became a highly potent delta-agonist (pEC(50), 9.90), slightly greater than deltorphin C (pEC(50), 9.56), with mu-agonism (pE(50), 7.57), while H-Dmt-Tic-Gly-NH-CH(2)-Bid (4) retained potent delta-antagonism (pA(2), 9.0) but with an order of magnitude less mu-agonism. Similarly, H-Dmt-Tic-Gly-NH-Ph (5) had nearly equivalent high delta-agonism (pEC(50), 8.52) and mu-agonism (pEC(50), 8.59), while H-Dmt-Tic-Gly-NH-CH(2)-Ph (6) whose spacer was longer by a single methylene group exhibited potent delta-antagonism (pA(2), 9.25) and very high mu-agonism (pEC(50), 8.57). These data confirm that the distance between the Dmt-Tic pharmacophore and a third aromatic nucleus is an important criterion in converting Dmt-Tic from a highly potent delta-antagonist into a potent delta-agonist or into ligands with mixed delta- and mu-opioid properties.     

Structure-activity relationships of dynorphin a analogues modified in the address sequence

The peptide [Pro3]Dyn A(1-11)-NH2 2 exhibits high affinity (K(i) = 2.4 nM) and over 2000-fold selectivity for the opioid receptor. Stepwise removal of the C-terminal residues from this ligand demonstrated that its positively charged Arg residues, particularly Arg6 and Arg7, were crucial for binding to the kappa receptor. Analogues shorter than seven amino acids lacked significant affinity for opioid receptors. Comparison with a series of truncated analogues of Dyn A showed that the relative losses in binding potency differed only slightly between the two series. The neutral residues Ile8 and Pro10 could be removed without significant loss in affinity for the kappa receptor. Their replacement, in the Pro3 analogue, with additional Arg residues led to analogues with improved kappa affinity (e.g., [Pro3,Arg8]Dyn A(1-11)-NH2 20: K(i)(kappa) = 0.44 nM). This type of modification did not compromise the high kappa selectivity of the Pro3 analogues. These findings support the view that a negatively charged domain in the putative second extracellular loop of the kappa receptor selectively recognizes residues 6-11 of dynorphin through electrostatic interactions. As with parent compound 2, analogue 20 and related compounds displayed kappa antagonist properties.     

A new structural motif for mu-opioid antagonists

On the basis of the structural features of the Dmt-Tic pharmacophore, a new motif leading to a fairly potent mu-opioid antagonist is described. This motif contains the 4-amino-1,2,4,5-tetrahydro-2-benzazepine-3-one skeleton as a substitute for the Tic residue, which provides the conformational constraint compatible with the mu-opioid receptor. The stereoselective synthesis of four stereoisomers is performed starting from homochiral 2',6'-dimethyltyrosine (Dmt) and o-aminomethylphenylalanine.     

Effect of N-alkyl and N-alkenyl substituents in noroxymorphindole, 17-substituted-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6,7:2',3'-indolomorphinans, on opioid receptor affinity, selectivity, and efficacy

The N-alkyl analogues (N-ethyl through N-heptyl), branched N-alkyl chain analogues (N-isopropyl, N-2-methylpropyl, and N-3-methylbutyl), and N-alkenyl analogues ((E)-N-3-methylallyl (crotyl), N-2-methylallyl, and N-3,3-dimethylallyl) were prepared in the noroxymorphindole series (17-substituted-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6,7:2',3'-indolomorphinans), and the effect of the N-substituent on opioid receptor affinity, selectivity, and efficacy was examined using receptor binding assays, [(35)S]GTPgammaS efficacy determinations, and smooth muscle functional assays (electrically stimulated mouse vas deferens and guinea pig ileum). All of the compounds acted as opioid antagonists, including those with N-substituents which usually confer either weak agonist-antagonist behavior (N-ethyl) or potent opioid agonist activity (N-pentyl) in morphinan-like ligands which interact with the mu-receptor. Several N-substituted noroxymorphindoles were found to be more mu/delta-selective than naltrindole (NTI). The N-2-methylallylnoroxymorphindole, in particular, was found to be more selective than NTI in receptor binding assays (mu/delta = 1700 vs 120; kappa/delta = 810 vs 140), as an antagonist in the GTPgammaS assay (mu/delta = 170 vs 140; kappa/delta = 620 vs 160), and considerably more selective than NTI in the functional assays (mu/delta > 2200 vs 90). It also had high affinity for the delta-opioid receptor (K(i) = 4.7 nM in the binding assay) and high antagonist potency (1.2 nM in the GTPgammaS assay; 8.9 nM in the MVD assay).     

Analgesic activity and opioid receptor selectivity of stereoisomers of ohmefentanyl isothiocyanate.

Ohmefentanyl is a very potent and highly selective agonist for mu-opioid receptors. We now study analgesia, in vitro activity and opioid receptor affinity of the stereoisomers of ohmefentanyl isothiocyanate. We found that some isomers of ohmefentanyl isothiocyanate had a potent analgesic effect and that all isomers except (3R,4S,2'S)-ohmefentanyl isothiocyanate had a more potent inhibitory action on the electrically evoked contractions of mouse vas deferens than of guinea pig ileum. The inhibitory actions could be antagonized by naloxone. However, compared with the activity of the corresponding stereoisomers of ohmefentanyl, these ohmefentanyl isothiocyanates had significantly reduced analgesia and in vitro activity. They also inhibited the binding of [3H]DPDPE ([D-Pen(2),D-Pen(5)]enkephalin) and [3H]DAGO ([D-Ala(2),Mephe(4),Gly-ol(5)]enkephalin) to opioid receptors in mouse brain membranes. The inhibitory effect of stereoisomers of ohmefentanyl isothiocyanate at mu-opioid receptors was markedly lower than that of their parent compounds. The affinity of stereoisomers of ohmefentanyl isothiocyanate for delta-opioid receptors was, however, greater than or equal to that of their corresponding stereoisomers of ohmefentanyl. The results showed that the introduction of an isothiocyanato group into the phenyl ring in position-1 of ohmefentanyl reduced bioactivity and affinity to mu-opioid receptors but that the selectivity of these compounds for delta-opioid receptors was enhanced. Isomer (3R,4S,2'R)-ohmefentanyl isothiocyanate showed highest selectivity for delta-opioid receptors (K(i)(mu)/K(i)(delta)=13.6) and potent analgesic activity (ED(50)=0.25 mg/kg).     

In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor, >10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.     

Nonpeptide analogues of dynorphin A(1-8): design, synthesis, and pharmacological evaluation of kappa-selective agonists

Two novel series of kappa opioid receptor agonist analogues of MPCB-GRRI and MPCB-RRI, hybrid ligands of MPCB ((-)-cis-N-(2-phenyl-2-carbomethoxy)cyclopropylmethyl-N-normetazocine ) and of the C-terminal fragments of dynorphin A(1-8), have been synthesized. The critical functional groups of the peptide fragments of hybrid compounds were maintained, and the binding affinities and selectivities for compounds 1-40 to mu, delta, and kappa opioid receptors were analyzed. Compounds 15 and 16, MPCB-Gly-Leu-NH-(CH(2))(n)()-NH-C(=NH)-C(4)H(9) (n = 5, 6), displayed high affinity and selectivity for kappa opioid receptors (K(i)(kappa) = 6.7 and 5.3 nM, K(i)(mu)/K(i)(kappa) = 375 and 408, and K(i)(delta)/K(i)(kappa) = 408 and 424, respectively). Since kappa agonists may also cause psychotomimetic effects by interaction with sigma sites, binding assays to sigma(1) sites were performed where compounds 15 and 16 showed negligible affinity (K(i) > 10 000). Compounds 15 and 16 were further characterized in vivo and showed potent antinociceptive activity in mouse abdominal constriction tests (ED(50) = 0.88 and 1.1 mg/kg, respectively), fully prevented by nor-BNI. Thus, these novel analogues open an exciting avenue for the design of peptidomimetics of dynorphin A(1-8).     

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).     

Activity profiles of dalargin and its analogues in mu-, delta- and kappa-opioid receptor selective bioassays.

1. To elucidate the structural features ensuring action of [D-Ala2, Leu5]-enkephalyl-Arg (dalargin), a series of dalargin analogues were tested for their effectiveness in depressing electrically-evoked contractions of the guinea-pig myenteric plexus-longitudinal muscle preparations (mu- and kappa-opioid receptors) and the vasa deferentia of the hamster (delta-opioid receptors), mouse (mu-, delta- and kappa-opioid receptors), rat (similar to mu-opioid receptors) and rabbit (kappa-opioid receptors). The naloxone KB values in the myenteric plexus were also obtained. 2. [L-Ala2]-dalargin was 19 times less potent than dalargin, and its pharmacological activity was peptidase-sensitive. The ratio of delta-activity to mu-activity for [L-Ala2]-dalargin was 6.78, and KB was 7.9 nM. This emphasizes the role that D-configuration of Ala2 plays in determining the active folding of dalargin molecule as well as in conferring resistance to peptidases. 3. [Met5]-dalargin was equipotent to dalargin in the myenteric plexus, but was more potent in the vasa deferentia of hamster and mouse (KB=5.5 nM). Leu5 and the interdependence of Leu5 and D-Ala2 are of importance for the selectivity of dalargin for mu-opioid receptors. 4. Dalarginamide was more potent and selective for mu-opioid receptors than dalargin, whilst dalarginethylamide, though equipotent to dalarginamide in the myenteric plexus, was more potent at delta-opioid receptors (KB=5.0 nM). [D-Phe4]-dalarginamide and N-Me-[D-Phe4]-dalarginamide were inactive indicating the contribution of L-configuration of Phe4 to the pharmacological potency of dalargin. 5. N-Me-[L-Phe4]-dalarginamide possessed the highest potency and selectivity for mu-opioid receptors (the ratio of delta-activity to mu-activity was 0.00053; KB=2.6 nM). The CONH2 terminus combined with the N-methylation of L-Phe4 increased the potency and selectivity of dalargin for mu-opioid receptors.     

Receptor binding and biological activity of the dermorphin analog Tyr-D-Arg(2)-Phe-Sar (TAPS)

The binding of Tyr-D-Arg(2)-Phe-sarcosine(Sar)(4) (TAPS), a proposed mu-opioid receptor-selective tetrapeptide analog of dermorphin to opioid receptors, was studied using selective binding assays for subtypes of mu-, delta- and kappa-opioid receptors. Subtype specific mu-opioid receptor binding was further characterized in the presence of sodium and guanosine nucleotides and the activity of TAPS in isolated guinea pig ileum was compared to other mu-opioid receptor-selective ligands. Further, the antinociceptive properties of TAPS following intrathecal (i.t.) administration in rats, as a model of spinal antinociception, were evaluated. The K(i)-values for TAPS at the mu(1)- and mu(2)-opioid receptor sites were 0.4 and 1.3 nM, respectively, suggesting high affinity binding to mu-opioid receptor binding sites with an increased selectivity to mu(1)-opioid receptor sites. The attenuated reduction of TAPS binding at the mu(2)-opioid receptor subtype in the presence of the stable guanosintriphosphate analog 5'-guanylylimidodiphosphate and sodium suggests a potential partial antagonist mode of action at this site.