Synthesis and Characterization of Selective Fluorescent Ligands for the neurokinin NK2 Receptor

Several fluorescent probes for the NK2 receptor were designed, synthesized, and pharmacologically characterized. These fluorescent ligands are analogues of the selective NK2 heptapeptide antagonist N-alpha-benzoyl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtained by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala1 and the subsequent coupling of the fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent derivatives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluorescein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and the fluorescent group was also studied. The most potent fluorescent antagonists were N-alpha-benzoyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pKi = 8.87 for NK2; N-alpha-benzoyl-Orn (delta-NBD)-Ala-D-Trp-Phe- D-Pro-Pro-Nle-NH2 (4B), pKi = 8.84; and N-alpha-benzoyl-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (3B), pKi = 8.83. These three compounds were highly selective for NK2 over NK3 and NK1 receptors. We show that these fluorescent ligands are useful tools for the detection of NK2 receptor expression by flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor interaction by spectrofluorimetry.