Design, synthesis and pharmacological characterization of a potent radioiodinated and photoactivatable peptidic oxytocin antagonist

Using a segment strategy, we have synthesized four iodinated photoactivatab le cyclic peptidic ligands of oxytocin, bearing a beta -mercapto-beta beta -cyclopentamethylene propionic group (Pmp) on their N-terminus. All the syn theses were RP-HPLC monitored, and the compounds were HPLC purified. They w ere characterized by IH NMR, MALDI-TOF, or FAB mass spectrometries. The aff inities of Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N(3))-NH2 (20), Pmp- Tyr-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N(3))-NH2 (21), Pmp-Tyr(Me)-Ile-Thr-Asn -Cys-Pro-Orn-Phe(3I,4N(3))-NH2 (22), and Pmp--Tyr-Ile-Thr-Asn-Cys-Pro-Orn-P he(3I,4N(3))-NH2 (23) were evaluated as inhibition constants (K-i, in nM) f or the human oxytocin receptor expressed in Chinese hamster ovary cells by displacement of a radioiodinated disulfide-cyclized antagonist (Elands et a l. Eur. J. Pharmacol. 1987, 147, 197-207). The most potent of them, compoun d 22, was synthesized by another method in order to allow its radiolabeling by I-125. Its dissociation constant (K-d) for the human oxytocin receptor, directly measured in saturation studies, was 0.25 +/- 0.04 nM, and its ant agonist properties were determined by inactivation of phospholipase C, thus obtaining an inactivation constant (K-inact) of 0.18 +/- 0.02 nM, evaluate d by inositol phosphate accumulation. This compound is a very good tool for the mapping of peptidic antagonist binding sites in the human oxytocin rec eptor.