SYNTHETIC LINEAR AND CYCLIC GLUCAGON ANTAGONISTS

The synthesis and biological activities of seven new glucagon analogue s are reported. The design of compounds 2-5 is based on potent antagon ists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concent rated specifically on modifications to histidine-1. In addition we hav e prepared two cyclic compounds 7 and 8, related to a linear in vivo a ntagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modification s involved substitution of His1 by the unnatural conformationally cons trained residue rahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic aci d (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg1 2,Lys17,18, Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12, Lys17,18, Glu 21]glucagon amide (3); zyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]gluca gon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18, Glu21]glucagon (5); [dHis1 ,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1) -[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the li near analogues, as expressed a percentage of glucagon binding, are 2.6 (2), 0.13 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nm, respectively (IC50 of glucagon = 1.3 nm). The IC50 Values for 8 at the high and low affinity sites a re 4.7 and 3451 nm, respectively. The cyclic analogues are characteriz ed by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables d ifferentiation of isomers of the cyclic glucagon analogues which diffe r only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 ar e glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual ami no acids which do not contain a primary alpha-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases. (C) Munksgaard 1 993.