'Carba'-carfentanil (trans isomer): a µ opioid receptor (MOR) partial agonist with a distinct binding mode.

There is strong evidence to indicate that a positively charged nitrogen of endogenous and exogenous opioid ligands forms a salt bridge with the Asp residue in the third transmembrane helix of opioid receptors. To further examine the role of this electrostatic interaction in opioid receptor binding and activation, we synthesized 'carba'-analogues of the highly potent µ opioid analgesic carfentanil (3), in which the piperidine nitrogen was replaced with a carbon. The resulting trans isomer (8b) showed reduced, but still significant MOR binding affinity (Ki(µ)=95.2nM) with no MOR versus DOR binding selectivity and was a MOR partial agonist. The cis isomer (8a) was essentially inactive. A MOR docking study indicated that 8b bound to the same binding pocket as parent 3, but its binding mode was somewhat different. A re-evaluation of the uncharged morphine derivative N-formylnormorphine (9) indicated that it was a weak MOR antagonist showing no preference for MOR over KOR. Taken together, the results indicate that deletion of the positively charged nitrogen in µ opioid analgesics reduces MOR binding affinity by 2-3 orders of magnitude and may have pronounced effects on the intrinsic efficacy and on the opioid receptor selectivity profile.

Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range of efficacies at the δ opioid receptor. Discovery of two highly potent and selective δ opioid agonists.

Analogues of the δ opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic=1,2,3,4-tetrahydroisoquinoline3-carboxylic acid) containing various 4'-[N-(alkyl or aralkyl)carboxamido]phenylalanine analogues in place of Tyr(1) were synthesized. The compounds showed subnanomolar or low nanomolar δ opioid receptor binding affinity and various efficacy at the δ receptor (antagonism, partial agonism, full agonism) in the [(35)S]GTPγS binding assay. Two analogues, [1-Ncp(1)]TIPP (1-Ncp=4'-[N-(2-(naphthalene-1-yl)ethyl)carboxamido]phenylalanine) and [2-Ncp(1)]TIPP (2-Ncp=4'-[N-(2-(naphthalene-2-yl)ethyl)carboxamido]phenylalanine), were identified as potent and selective δ opioid agonists.

Blood-brain barrier penetration by two dermorphin tetrapeptide analogues: role of lipophilicity vs structural flexibility.

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

Dicarba analogues of the cyclic enkephalin peptides H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH(2) retain high opioid activity.

Dicarba analogues of the cyclic opioid peptides H-Tyr-c[d-Cys-Gly-Phe-d(or l)-Cys]NH2 were synthesized on solid phase by substituting allylglycines for the cysteines and cyclization by ring-closing metathesis between the side chains of the allylglycine residues. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated -CH2-CH2- bridged peptides. The dicarba analogues retained high mu and delta agonist potencies. Remarkably, the trans isomer of H-Tyr-c[d-Allylgly-Gly-Phe-l-Allylgly]NH2 was a mu agonist/delta agonist with subnanomolar potency at both receptors.

Synthesis and characterization of potent and selective mu-opioid receptor antagonists, [Dmt(1), D-2-Nal(4)]endomorphin-1 (Antanal-1) and [Dmt(1), D-2-Nal(4)]endomorphin-2 (Antanal-2).

To synthesize potent antagonists of the mu-opioid receptor, we prepared a series of endomorphin-1 and endomorphin-2 analogues with 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal) in position 4. Some of these analogues displayed weak antagonist properties. We tried to strengthen these properties by introducing the structurally modified tyrosine residue 2,6-dimethyltyrosine (Dmt) in place of Tyr1. Among the synthesized compounds, [Dmt1, d-2-Nal4]endomorphin-1, designated antanal-1, and [Dmt1, d-2-Nal4]endomorphin-2, designated antanal-2, turned out to be highly potent and selective mu-opioid receptor antagonists, as judged on the basis of two functional assays, the receptor binding assay and the hot plate test of analgesia. Interestingly, another analogue of this series, [Dmt1, d-1-Nal4]endomorphin-1, turned out to be a moderately potent mixed mu-agonist/delta-antagonist.

Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice.

The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure-activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions.

Replacement of the N-terminal tyrosine residue in opioid peptides with 3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid (Dcp) results in novel opioid antagonists.

3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist.

Cyclic dermorphin tetrapeptide analogues obtained via ring-closing metathesis.

The dermorphin-derived cyclic tetrapeptide analogues H-Tyr-c[D-Cys-Phe-Cys]NH(2) and H-Tyr-c[D-Cys-Phe-D-Cys]NH(2) are opioid agonists at the mu and delta receptor. To enhance the metabolic stability of these peptides, we replaced the disulfide bridge with a bis-methylene moiety. This was achieved by solid-phase synthesis of the linear precursor peptide containing allylglycine residues in place of the Cys residues, followed by ring-closing metathesis. In the case of the peptide with L-configuration in the 4-position both the cis and the trans isomer of the resulting olefinic peptides were formed, whereas the cis isomer only was obtained with the peptide having the D-configuration in position 4. Catalytic hydrogenation yielded the saturated -CH(2)-CH(2)- bridged peptides. In comparison with the cystine-containing parent peptides, all olefinic peptides showed significantly reduced mu and delta agonist potencies in the guinea pig ileum and mouse vas deferens assays. The -CH(2)-CH(2)-bridged peptide with l-configuration in the 4-position was equipotent with its cystine-containing parent in both assays, whereas the bis-methylene analogue with D-configuration in position 4 was 10-27-fold less potent compared to its parent. The effect of the disulfide replacements with the -CH=CH- and -CH(2)-CH(2)- moieties on the conformational behavior of these peptides was examined by theoretical conformational analysis which provided plausible explanations in terms of structural parameters for the observed changes in opioid activity.

Synthesis and biological evaluation of compact, conformationally constrained bifunctional opioid agonist - neurokinin-1 antagonist peptidomimetics.

A reported mixed opioid agonist - neurokinin 1 receptor (NK1R) antagonist 4 (Dmt-D-Arg-Aba-Gly-(3',5'-(CF3)2)NMe-benzyl) was modified to identify important features in both pharmacophores. The new dual ligands were tested in vitro and subsequently two compounds (lead structure 4 and one of the new analogues 22, Dmt-D-Arg-Aba-ß-Ala-NMe-Bn) were selected for in vivo behavioural assays, which were conducted in acute (tail-flick) and neuropathic pain models (cold plate and von Frey) in rats. Compared to the parent opioid compound 33 (without NK1R pharmacophore), hybrid 22 was more active in the neuropathic pain models. Attenuation of neuropathic pain emerged from NK1R antagonism as demonstrated by the pure NK1R antagonist 6. Surprisingly, despite a lower in vitro activity at NK1R in comparison with 4, compound 22 was more active in the neuropathic pain models. Although potent analgesic effects were observed for 4 and 22, upon chronic administration, both manifested a tolerance profile similar to that of morphine and cross tolerance with morphine in a neuropathic pain model in rat.

Highly potent fluorescent analogues of the opioid peptide [Dmt1] DALDA.

H-Dmt-D-Arg-Phe-Lys-NH2 (Dmt=2',6'-dimethyltyrosine) ([Dmt1] DALDA) is a highly potent and selective micro opioid peptide agonist capable of producing an antinociceptive effect after systemic administration. Fluorescent analogues of [Dmt1] DALDA containing either beta-dansyl-L-alpha,beta-diaminopropionic acid [Dap(dns)] or beta-anthraniloyl-L-alpha,beta-diaminopropionic acid [Dap(atn)] in place of Lys4 were synthesized. Both analogues retained subnanomolar mu opioid receptor binding affinity, very high mu opioid agonist activity in the guinea pig ileum assay and extraordinarily high antinociceptive activity in the mouse tail-flick test (intrathecal administration). The maxima of the fluorescence emission spectra recorded in Tris-HCl buffer (pH 6.6) indicated a completely aqueous environment of the fluorophore in both peptides. The high fluorescence quantum yield (phi=0.358) of the [Dap(atn)4] analogue was particularly remarkable. These fluorescent [Dmt1] DALDA analogues represent valuable pharmacological tools for various applications, including studies on the binding to receptors and other biopolymers, cellular uptake and intracellular distribution, and tissue distribution.

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists.

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.

Bifunctional µ/δ opioid peptides: variation of the type and length of the linker connecting the two components.

On the basis of evidence that opioid compounds with a mixed µ agonist/δ antagonist profile may produce an antinociceptive effect with low propensity to induce side effects, bifunctional opioid peptides containing the µ agonist H-Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1) ]DALDA; Dmt = 2',6'-dimethyltyrosine) connected tail-to-tail via various α,ω-diaminoalkyl- or diaminocyclohexane linkers to the δ antagonists H-Tyr-TicΨ[CH(2) -NH]Cha-Phe-OH (TICP[Ψ]; Cha = cyclohexylalanine, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), H-Dmt-Tic-OH or H-Bcp-Tic-OH (Bcp = 4'-[N-((4'-phenyl)phenethyl)carboxamido]phenylalanine) were synthesized and pharmacologically characterized in vitro. Bifunctional [Dmt(1) ]DALDA→NH-(CH(2) )(n) -NH←TICP[Ψ] compounds (n = -12) showed decreasing µ and δ receptor binding affinities with increasing linker length. As expected, several of the bifunctional peptides were µ agonist/δ antagonists with low nanomolar µ and δ receptor binding affinities. However, compounds with unexpected opioid activity profiles, including a µ partial agonist/δ partial agonist, µ antagonist/δ antagonists and µ agonist/δ agonists, were also identified. These results indicate that the binding affinities and intrinsic efficacies of these bifunctional compounds at both receptors depend on the length and type of the linker connecting the µ and δ components. An important recommendation emerging from this study is that the in vitro activity profiles of bifunctional compounds containing an agonist and an antagonist component connected via a linker need to be determined prior to their pharmacological evaluation in vivo.

Variation of the net charge, lipophilicity, and side chain flexibility in Dmt(1)-DALDA: Effect on Opioid Activity and Biodistribution.

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic µ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced µ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

N-methylated cyclic enkephalin analogues retain high opioid receptor binding affinity.

In an effort to improve the bioavailability of the non-selective, cyclic enkephalin analogues H-Dmt-c[d-Cys-Gly-Phe-d(or L)-Cys]NH(2) (Dmt = 2',6'-dimethyltyrosine), analogues N-methylated at the Phe(4) and/or Cys(5) residue were synthesized. In comparison with the non-methylated parent peptides, all mono- and N-di-methylated analogues in general retained high binding affinities at all three opioid receptors and high opioid agonist potencies in functional opioid activity assays. The results indicate that the progressive conformational restriction in these compounds upon mono- and di-N-methylation did not significantly affect the in vitro opioid activity profile. A low-energy conformer identified for the conformationally most restricted analogue of the series, H-Dmt-c[D-Cys-Gly-Phe(NMe)-L-Cys(NMe)]NH(2) (6), showed good spatial overlap of the essential pharmacophoric moieties with those in the proposed mu receptor-bound conformation of the mu-selective opioid peptide JOM-6 [H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH(2)] (Pen = penicillamine) [Mosberg M.I. and Fowler C.B. (2002) J Peptide Res; 60:329-335], in agreement with the moderate mu selectivity determined for this compound. An analogue of 6 containing (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Dmt(1) was an opioid antagonist with quite high opioid receptor binding affinities and can be expected to show improved bioavailability because of its further increased lipophilicity and reduced hydrogen-bonding capacity.

"Carba"-analogues of fentanyl are opioid receptor agonists.

There is evidence to indicate that the Asp residue in the third transmembrane helix (TMH) of opioid receptors forms a salt bridge with the positively charged nitrogen of endogenous and exogenous opioid ligands. To further examine the role of this electrostatic interaction in receptor binding and activation, we synthesized "carba"-analogues of a published fentanyl analogue containing a 3-(guanidinomethyl)-benzyl group in place of the phenyl moiety attached to the ethylamido group (C. Dardonville et al., Bioorg. Med. Chem. 2006, 14, 6570-6580 (1)), in which the piperidine ring nitrogen was replaced with a carbon. As expected, the resulting cis and trans isomers (8a and 8b) showed reduced mu and kappa opioid receptor binding affinities as compared to 1 but, surprisingly, retained opioid full agonist activity with about half the potency of leucine-enkephalin in the guinea pig ileum assay. In conjunction with performed receptor docking studies, these results indicate that the electrostatic interaction of the protonated nitrogen in the piperidine ring of fentanyl analogues with the Asp residue in the third TMH is not a conditio sine qua non for opioid receptor activation.

Agonist vs antagonist behavior of delta opioid peptides containing novel phenylalanine analogues in place of Tyr(1).

The novel phenylalanine analogues 4'-[N-((4'-phenyl)phenethyl)carboxamido]phenylalanine (Bcp) and 2',6'-dimethyl-4'-[N-((4'-phenyl)phenethyl)carboxamido]phenylalanine (Dbcp) were substituted for Tyr(1) in the delta opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic = tetrahydroisoquinoline-3-carboxylic acid). Unexpectedly, [Bcp(1)]TIPP was a potent, selective delta opioid agonist, whereas [Dbcp(1)]TIPP retained high delta antagonist activity. Receptor docking studies indicated similar binding modes for the two peptides except for the biphenylethyl moiety which occupied distinct receptor subsites. The dipeptide H-Dbcp-Tic-OH was a highly selective delta antagonist with subnanomolar delta receptor affinity.

Cyclic opioid peptide agonists and antagonists obtained via ring-closing metathesis.

The opioid peptide H-Tyr-c[D-Cys-Phe-Phe-Cys]NH(2) cyclized via a methylene dithiother is a potent and selective mu opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2',6'-dimethyltyrosine (Dmt), 3-[2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in the 1-position were prepared. The peptides were synthesized on solid-phase by substituting d-allylglycine and (2S)-2-amino-5-hexenoic acid in position 2 and 5, respectively, followed by ring-closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated -CH(2)-CH(2)- bridged peptides. All six Tyr(1)- and Dmt(1)-dicarba analogues retained high mu and delta opioid agonist potency and showed only slight or no preference for mu over delta receptors. As expected, the six Dhp(1)- and (2S)-Mdp(1)-dicarba analogues turned out to be mu opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the delta receptor. The obtained results indicate that the mu versus delta receptor selectivity and the efficacy at the delta receptor of these cyclic peptides depend on distinct conformational characteristics of the 15-membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe(3) and Phe(4) side chains.

Potent opioid peptide agonists containing 4'-[N-((4'-phenyl)-phenethyl)carboxamido]phenylalanine (Bcp) in place of Tyr.

Analogues of the opioid peptides H-Tyr-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 (non-selective), H-Tyr-D-Arg-Phe-Lys-NH2 (mu-selective) and dynorphin A(1-11)-NH2 (kappa-selective) containing 4'-[N-((4'-phenyl)-phenethyl)carboxamido]phenylalanine (Bcp) in place of Tyr1 were synthesized. All three Bcp1-opioid peptides retained high mu opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr1-containing parent peptides. The cyclic peptide HBcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 turned out to be an extraordinarily potent, mu-selective opioid agonist, whereas the Bcp1-analogue of dynorphin A(1-11)-NH2 displayed partial agonism at the mu receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand's ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.