Structure-activity relations of a series of analogues of the two endo­genous morphine-like peptides, leucine-enkephalin and methionine-enkephalin are examined on the basis of ( a ) effects on the mouse vas deferens and the guinea pig ileum and ( b ) affinities for the rat brain opiate receptor. In the mouse vas deferens, metabolism of the peptides by proteolysis is not a major influence on activity. In contrast, however, brain opiate re­ceptor preparations contain an abundance of proteolytic enzymes, the effects of which can be minimized by conducting opiate receptor binding assays at 0 °C and in the presence of bacitracin. The potentiation of biological activity and opiate receptor binding affinity by replacing the Gly 2 residue in the natural enkephalins by d-Ala, is discussed both in terms of increased stability of the Tyr-d-Ala bond to aminopeptidases and of the stabilization of the peptide conform­ation as present in the receptor-peptide complex. The substitution of the Leu 5 - or Met 5 -residue by the corresponding d-amino acid contributes little to proteolytic stability, which emphasizes that the predominating site at which metabolism occurs is the Tyr 1 -Gly 2 bond. Of the analogues described, [d-Ala 2 , d-Leu 5 ]-enkephalin is the most active peptide in the three assay systems, the mouse vas deferens, the guinea pig ileum and the rat brain opiate receptor preparations. Substitutions by the respective d-amino acids d-Tyr and d-Phe at positions 1 and 4 reduce both the potency and binding affinity and emphasize the importance of stereochemical acceptability at these positions. The promotion of receptor binding by d-amino acids is examined, particularly with respect to implied peptide conformations. The experi­mental data have been analysed for the relative influence of metabolic and conformational factors.

Leucine‐enkephalin and some analogues were assayed for activity in vitro on the mouse vas deferens and for binding to opiate receptors from rat brain. The experimental data were analysed in terms of the stringency for glycine, a D‐amino acid or an L‐amino acid at each position in the peptide. The observed configurational specificity was compared with the stringency that would be predicted to occur if enkephalin adopted certain hydrogen‐bonded conformations at the receptor. A small subset of the conformations examined was found to be compatible with the experimental data.

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1 To investigate peripherally mediated antinociceptive effects of opioids, the activity of a novel polar enkephalin analogue H-Tyr-D-Arg-Gly-Phe (4-NO2)-Pro-NH2 (BW443C) has been compared with those of classical tertiary opiates against different nociceptive stimuli in the mouse. 2 In chemically-induced writhing models BW443C, administered subcutaneously, demonstrated dose-related antinociceptive effects less potent than morphine and of a similar order to pethidine and D-propoxyphene. In assays using heat as the noxious stimulus BW443C was markedly less potent than any of the opiates tested. 3 In heat-induced assays, but not in chemically-induced writhing assays, BW443C demonstrated a 'U'-shaped dose-time response relationship. Morphine, pethidine and D-propoxyphene showed simple, approximately linear, dose-time effects in all assays. 4 When given subcutaneously, the inhibitory effects of BW443C and morphine in chemicallyinduced writhing were antagonized by naloxone given intraperitoneally. The inhibitory effects on writhing of BW443C, but not those of morphine, were also antagonized by prior intraperitoneal administration of the quaternary opioid antagonist N-methyl nalorphine. When this antagonist was administered intracerebroventricularly, the antinociceptive effects in writhing of both BW443C and morphine were antagonized. 5 It is concluded that BW443C, being only poorly able to cross the blood brain barrier, demonstrates peripherally mediated opioid antinociceptive effects in chemically-induced writhing models. In heatinduced models, that detect centrally acting opioids, BW443C is effective only at high doses and at time intervals after dosing sufficient to allow slow penetration of drug into the CNS. It is suggested that the peripheral antinociceptive actions of BW443C are mediated by inhibition of sensory neurones.

The design, synthesis, and biological activity of a series of highly polar enkephalin-related pentapeptides are reported. These analogues incorporate structural features that exclude them from the central nervous system and thereby restrict their action to peripherally located receptors. Hydrophilic analogues were obtained by introduction of polar D-amino acid residues at position 2 and, in certain cases, by conversion of the N-terminal amino group of the Tyr residue to a guanidino function. The peptides were synthesized by classical solution methods. All compounds demonstrated in vitro opioid activity in the GPI and all were shown to possess antinociceptive activity in chemically induced writhing models. The analgesic effects were shown to be predominantly peripherally mediated by antagonism of antinociception with the peripheral antagonist N-methylnalorphine. Comparative data obtained in writhing and hot-plate tests were also supportive of a peripheral mode of action. Compound 13a, L-tyrosyl-D-arginylglycyl-L-4-nitrophenylalanyl-L-prolinamide (BW 443C), was identified as having a favorable pharmacological profile, indicating a high level of peripheral selectivity, and worthy of further investigation.