'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.

N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/µ partial agonist activity. Guanidinylation of the mixed µ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed µ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed µ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.

Equipotent enantiomers of cyclic opioid peptides at µ opioid receptor.

Head-to-tail cyclized analogues of the µ opioid receptor (MOR) agonist tetrapeptides DALDA (H-Tyr-D-Arg-Phe-Lys-NH2 and [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) and their enantiomers (mirror-image isomers) were synthesized and pharmacologically characterized in vitro. Three pairs of enantiomeric cyclic peptides with both mirror-image isomers having equipotent MOR binding affinities but different binding affinities at the δ and κ opioid receptors were identified. The cyclic peptide enantiomers c[-D-Arg-Phe-Lys-Tyr-] (1) and c[-Arg-D-Phe-D-Lys-D-Tyr-] (2) showed nearly identical MOR binding affinity (1 - 2 nM) and equipotent MOR antagonist activity. The results of a MOR docking study indicated a very similar binding mode of the two enantiomers with nearly complete spatial overlap of the peptide ring structures and side chain interactions with the same MOR residues. Compounds 1 and 2 represent the first pair of enantiomeric G-protein-coupled receptor (GPCR) ligands having multiple chiral centers, with both optical antipodes showing equal, low nanomolar receptor binding affinity.

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.

A Cyclic Tetrapeptide ("Cyclodal") and Its Mirror-Image Isomer Are Both High-Affinity µ Opioid Receptor Antagonists.

Head-to-tail cyclization of the µ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2',6'-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) ("cyclodal"), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood-brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity.

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.

"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.

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.

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.

Novel opioid peptide derived antagonists containing (2S)-2-methyl-3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid [(2S)-Mdcp].

A synthesis of the novel tyrosine analogue (2 S)-2-methyl-3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid [(2 S)-Mdcp] (15) was developed. In (2 S)-Mdcp, the amino and hydroxyl groups of 2',6'-dimethyltyrosine are replaced by a methyl and a carbamoyl group, respectively, and its substitution for Tyr (1) in opioid agonist peptides resulted in compounds showing antagonism at all three opioid receptors. The cyclic peptide (2 S)-Mdcp-c[D-Cys-Gly-Phe(pNO 2)-D-Cys]NH 2 (1) was a potent and selective mu antagonist, whereas (2 S)-Mdcp-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH (3) showed subnanomolar delta antagonist activity and extraordinary delta selectivity.

A novel cyclic enkephalin analogue with potent opioid antagonist activity.

2',6'-Dimethyl substitution of the Tyr(1) residue in opioid agonist peptides and deletion of the N-terminal amino group, as achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp), have been shown to produce opioid antagonists. To examine the effect of beta-methylation of Dhp(1) in opioid peptides on the activity profile, stereoselective syntheses of (3S)- and (3R)-3-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(3S)- and (3R)-Mdp] were carried out. In comparison with the cyclic parent antagonist peptide Dhp-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2), the methylated analogue (3S)-Mdp-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) showed higher micro, delta and kappa antagonist potencies in functional assays and higher binding affinities for micro, delta and kappa opioid receptors (K(i)(micro)=2.03 nM; K(i)(delta)=2.34 nM; K(i)(kappa)=49.5 nM), whereas the corresponding (3R)-Mdp(1)-analogue was less potent by 1-2 orders of magnitude.