Pseudopeptide Analogues of Substance P and Leucine Enkephalinamide Containing the Ψ(CH₂O) Modification: Synthesis and Biological Activity

The isosteric methyleneoxy Ψ(CH₂O) function was employed as a novel peptide-bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu⁶,Phe⁸Ψ(CH₂O)Gly⁹]SP_6–11 (7) of SP related C-terminal hexapeptide [pGlu⁶]SP_6–11 and two pseudopeptide analogues of [Leu⁵]enkephalinamide, [Tyr¹Ψ(CH₂O)Gly²,Leu⁵]enkephalinamide (11) and [Gly²Ψ(CH₂O)- Gly³,Leu⁵]enkephalinamide (17), were synthesized. The N^α-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC₅₀ = 4.8 nM) of substance P as compared to the parent peptide [pGlu⁶]SP_6–11 (EC₅₀ = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC₅₀(NK-1)/EC₅₀(NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu⁶]SP_6–11, EC₅₀(NK-1)/EC₅₀(NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC₅₀ = 2 μM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC₅₀, = 2.3 μM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the μ and δ binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC₅₀ = 30 nM), being equipotent to [D-Ala²,Leu⁵]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC₅₀ = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both the μ and δ binding sites with increased selectivity for the i sites as shown by the ratio of the apparent affinities for both receptors, K_i(δ)/K_i(μ) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed.