Opioid compounds with mixed mu agonist/delta antagonist properties are expected to be analgesics with low propensity to produce tolerance and dependence. In an effort to strengthen the mu agonist component of the mixed mu agonist/delta antagonist H-Tyr-Tic-Phe-Phe-NH(2) (TIPP-NH(2)), analogues containing structurally modified tyrosine residues in place of Tyr(1) were synthesized. Among the prepared compounds, H-Dmt-Tic-Phe-Phe-NH(2) (DIPP-NH(2); Dmt = 2',6'-dimethyltyrosine) and H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2) (DIPP-NH(2)[Psi]) retained a mixed mu agonist/delta antagonist profile, as determined in the guinea pig ileum and mouse vas deferens assays, whereas H-Tmt-Tic-Phe-Phe-NH(2) (Tmt = N,2',6'-trimethyltyrosine) was a partial mu agonist/delta antagonist and H-Tmt-TicPsi[CH(2)NH]Phe-Phe-NH(2) was a mu antagonist/delta antagonist. DIPP-NH(2)[Psi] showed binding affinities in the subnanomolar range for both mu and delta receptors in the rat brain membrane binding assays, thus representing the first example of a balanced mu agonist/delta antagonist with high potency. In the rat tail flick test, DIPP-NH(2)[Psi] given icv produced a potent analgesic effect (ED(50) = 0.04 microg), being about 3 times more potent than morphine (ED(50) = 0.11 microg). It produced less acute tolerance than morphine but still a certain level of chronic tolerance. Unlike morphine, DIPP-NH(2)[Psi] produced no physical dependence whatsoever upon chronic administration at high doses (up to 4.5 microg/h) over a 7-day period. In conclusion, DIPP-NH(2)[Psi] fulfills to a large extent the expectations based on the mixed mu agonist/delta antagonist concept with regard to analgesic activity and the development of tolerance and dependence.

Opioid peptide analogs consisting entirely of aromatic amino acid residues and contining conformationally restricted phenylalanine derivatives in position 2 of the peptide sequence were synthesized and pharmacologically characterized in vitro. Whereas the existence of at least three major opioid receptor classes (u, 6, and K) is now well-established, the development ofpotent opioid agonists and antagonists with high specificity for each receptor type and of ligands with receptor-specific agonist/antagonist properties continues to be an important goal in opioid pharmacology. The fact that A and 8 opioid receptors differ from one another in their conformational requirements for peptide ligands was first established through comparison of the receptor binding profiles of a cyclic enkephalin analog and its linear correlate (1). This observation led to the realization that conformational restriction of peptides either locally through incorporation of backbone or side-chain conformational constraints at a specific amino acid residue or more globally through peptide cyclizations may often result in improved receptor selectivity. The use of this strategy resulted in a number of conformationally restricted opioid peptide analogs with agonist properties that showed high preference for either 1L or 6 receptors (for a review, see ref.2). It has often been speculated but never demonstrated unambiguously that conformational restriction of peptides in some cases might also reduce or even totally abolish their intrinsic activity ("efficacy") and, thus, may produce partial agonists or antagonists. No examples of opioid peptide analogs with significant antagonist properties as a consequence of conformational restriction have been reported to date. The only opioid-peptide-derived antagonists with reasonable potency described so far were obtained through diallylation of the N-terminal amino group. An enkephalin analog of this type, NN-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864; Aib = aminoisobutyric acid) (3), has been useful as a 6-selective antagonist.In this paper we report that the tetrapeptide amide H-Tyr-D-Phe-Phe-Phe-NH2 (la) is a potent p-selective opioid agonist. This compound consists entirely of aromatic amino acids that can be conformationally restricted in a number of interesting ways. We show that substitution of the D and L isomers of the conformationally restricted phenylalanine analogs Na-methylphenylalanine (NMePhe) and tetrahydro-3-isoquinoline carboxylic acid (Tic) (Fig. 1) for D-Phe2 in peptide la produced astonishing changes in receptor affinities and intrinsic activities. Most importantly, these structureactivity studies defined a class of potent and selective 6 antagonists, characterized by the N-terminal sequence H-Tyr-Tic-Phe-. MATERIALS AND METHODSPeptide Synthesis. Peptide analogs 1-7 were synthesized by the usual solid-phase technique with N"-t-butyloxycarbonylprotected amino acids and with benzotriazol-1-yl-oxytris-(dimethylamino)phosphonium hexafluorophosphate as coupling agent as described el...

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Recent studies showed that dermorphin and enkephalin analogues containing two methyl groups at the 2',6'-positions of the Tyr(1) aromatic ring and lacking an N-terminal amino group were moderately potent delta and mu opioid antagonists. These results indicate that a positively charged N-terminal amino group may be essential for signal transduction but not for receptor binding and suggested that its deletion in agonist opioid peptides containing an N-terminal 2',6'-dimethyltyrosine (Dmt) residue may represent a general way to convert them into antagonists. In an attempt to develop dynorphin A (Dyn A)-derived kappa opioid antagonists, we prepared analogues of [Dmt(1)]Dyn A(1-11)-NH2 (1), in which the N-terminal amino group was either omitted or replaced with a methyl group. This was achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp]. Compounds were tested in the guinea pig ileum and mouse vas deferens bioassays and in rat and guinea pig brain membrane receptor binding assays. All analogues turned out to be potent kappa antagonists against Dyn A(1-13) and the non-peptide agonist U50,488 and showed only weak mu and delta antagonist activity. The most potent and most selective kappa antagonist of the series was [(2S)-Mdp(1)]Dyn A(1-11)-NH2 (5, dynantin), which showed subnanomolar kappa antagonist potency against Dyn A(1-13) and very high kappa selectivity both in terms of its K(e) values determined against kappa, mu, and delta agonists and in terms of its ratios of kappa, mu, and delta receptor binding affinity constants. Dynantin is the first potent and selective Dyn A-derived kappa antagonist known and may complement the non-peptide kappa antagonists norbinaltorphimine and GNTI as a pharmacological tool in opioid research.

Pseudopeptide analogues of the delta opioid antagonists H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic-Phe-OH (TIP) containing a reduced peptide bond between the Tic2 and Phe3 residues were synthesized. The two compounds, H-Tyr-Tic psi [CH2NH]Phe-Phe-OH (TIPP [psi]) and H-Tyr-Tic psi-[CH2NH]Phe-OH (TIP [psi]), were tested in mu-, delta-, and kappa-receptor-selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. In comparison with their respective parent peptides, both pseudopeptide analogues showed increased delta antagonist potency in the MVD assay, higher delta receptor affinity and further improved delta receptor selectivity. The more potent compound, TIPP [psi], displayed subnanomolar delta receptor affinity and in direct comparisons with other selective delta ligands was shown to have unprecedented delta specificity (Ki mu/Ki delta = 10,500). Furthermore, this compound turned out to be highly stable against enzymatic degradation and, unlike other delta antagonists, showed no mu or kappa antagonist properties. TIPP [psi] is likely to find wide use as a pharmacological tool in opioid research.

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In an effort to determine the effect of side chain conformational restriction on opioid receptor selectivity, the cyclic phenylalanine analogues 2-aminoindan-2-carboxylic acid (Aic), 2-aminotetralin-2-carboxylic acid (Atc), and tetrahydroisoquinoline-3-carboxylic acid (Tic) were substituted for Phe in the potent cyclic opioid peptide analogue H-Tyr-D-Orn-Phe-Glu-NH2, which lacks significant opioid receptor selectivity. Compounds were tested in mu- and delta-opioid receptor representative binding assays and bioassays in vitro. The analogue H-Tyr-D-Orn-Aic-Glu-NH2 was found to be a potent agonist with high preference of mu receptors over delta receptors. Opening of the five-membered ring of Aic in the latter peptide, as achieved through substitution of C alpha-methylphenylalanine or o-methylphenylalanine, resulted in only slightly selective compounds, indicating that the high mu selectivity of the Aic analogue is exclusively the consequence of the imposed side chain conformational restriction. Both diastereoisomers of H-Tyr-D-Orn-(D,L)-Atc-Glu-NH2 were highly mu-selective and, in contrast to the weak affinity observed with the D-Phe3 analogue as compared to the L-Phe3 analogue, both had similar potency. Thus, stereospecificity was lost as a consequence of side chain conformational restriction. Further structure-activity data obtained with analogues containing L- or D-homophenylalanine (Hfe) or 3-(1'-naphthyl)alanine (Nap) in place of Phe3 and consideration of geometric interrelationships between Nap and the L and D isomers of Atc, Hfe, and Phe led to the proposal that the D-Phe3 and the D-Atc3 analogue may have different modes of binding to the receptor. The very low potency observed with H-Tyr-D-Orn-N alpha MePhe-Glu-NH2 (N alpha MePhe = N alpha-methylphenylalanine) and H-Tyr-D-Orn-Tic-Glu-NH2 indicated that N alpha-alkylation at the 3-position is detrimental to activity.