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Through design, synthesis, and biological testing of constrained gonadotropin releasing hormone (GnRH) antagonists, we are studying the structural requirements for biological activity. Here we describe the conformational analysis in solution of a highly potent, dicyclic GnRH antagonist, dicyclo(4-10/5,5‘-8)[Ac-d-2Nal1,d-pClPhe2,d-3Pal3,Asp4,Glu5(Gly),d-Arg6,Dbu8,Dpr10]GnRH (1), using NMR spectroscopy. The dicyclic part of this molecule adopts a preferred conformation containing a type II β turn around residues 5−6, nested with a type I‘ β turn around residues 6−7, and a type II β-turn-like structure involving residue 9 and the side chain of residue 10, which is stabilized by hydrogen bonds between Leu7 NH/Asp4 CO, Dbu8 NHδ/Glu5 CO, and Dpr10 NHγ/Dbu8 CO. This is a novel conformation that had not been observed previously in any constrained GnRH antagonist and is remarkably different from that found for another dicyclic (4-10/5-8) GnRH antagonist with very similar sequence, dicyclo(4-10/5-8)[Ac-d-2Nal1,d-pC...

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.