Eight new dermorphin tetrapeptides, X-Tyr-D-MetO-Phe-aa-Y (X = H, H2N = C(NH); aa = Gly, 2-aminoethanol, sarcosine; Y = NH2, NH-alkyl), were prepared and tested for opioid activity. They show dose-related naloxone-reversible opioid effects in vitro and in vivo. H-Tyr-D-MetO-Phe-Gly-NH2 (I) (guinea pig ileum IC50 = 13.6 nM; tail-flick ED50 = 1.97 pmol/mouse, icv, and 0.65 mumol/kg, sc), though less effective in the periphery, has central activities higher than those of dermorphin H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2. Following intracerebroventricular or subcutaneous administrations in mice, I is about respectively 1500 and 17 times as potent an analgesic as morphine.

We studied the effect of partial retro-inverso modification of selected peptide bonds of N-terminal tetrapeptide analogues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2). Among the 14 compounds synthesized and tested for opioid activity, some tetrapeptides have the C-terminus carrying different amide moieties; retromodifications concern the Phe-Gly bond (Ia-f) and/or the C-terminal carboxamide function (IIIa-d, IIa-d). All pseudotetrapeptide derivatives showed opioid activity in vitro and in vivo. The most potent compounds (II) have a biological potency comparable with that of the original tetrapeptides in the guinea pig ileum preparation and in the mouse tail-flick test after intracerebral or subcutaneous administration.

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SynopsisThe synthesis in solution of some modified (D)Ala2,Leu5-enkephalins has been carried out. The lipophilic properties of the parent compound have been modified by amidation of the carboxyl function with alkylamines of increasing hydrophilicity to increase permeability of the blood-brain barrier. Attempts to reduce enzymatic degradation have been carried out either by reductive glucosamination or by amidation of the carboxyl function with 2-amino-2-deoxy-~-D-glucopyranose. Esterification of the carboxyl function of (D)Ala2,Leu5-enkephalin with polyethylenglycole lo00 has also been carried out. The effects induced by these modifications have been evaluated by in vitro and in uiuo tests.Enkephalins (Tyr-Gly-Gly-Phe-X, X = Met or Leu)l are the smallest members of a family of endogenous peptides with opioidlike activity and have been thoroughly investigated in the past few years. Structure-activity relationships indicate that substitution of an amide group a t the carboxyl terminus and D-alanine at the second position enhance the potency of Leu-enkephalin ( L e~~-E n k ) .~ Stabilization of a 0-turn involving residues in positions 2 and 33 and protection against proteolytic degradation offered by D-alanine and by the C-terminal amide group have been offered as explanation for the enhanced potency of (~)Ala~,Leu~-EnkNHz.We report the synthesis of two (~) A l a~, L e u~-E n k N-alkylated amides, by conventional procedures with isolation and characterization of intermediates and final products. Enhancement of lipophilicity of (D)-Ala2,Leu5-EnkNH2 by modification a t the fifth residue, which is not essential for biological a~t i v i t y ,~ should increase permeability through the blood-brain barrier.Moreover, we tried to introduce features into the (D)Ala2,Leu5-enkephalin molecule that further retard enzymatic degradation but still retain

H-Tyr-D-Ala-PheGly-Tyr-Pro-Ser-NH2 (demorphin), an opiate-like peptide, and tri-, tetra-, penta-and hexapeptide-amide analogs, were synthesized by conventional methods in solution, to determine the minimum peptide chainlength, required for analgesic activity.