Design of potent dicyclic (1-5/4-10) gonadotropin releasing hormone (GnRH)antagonists

In three earlier papers, the structures and biological potencies of numerou s mono- and dicyclic antagonists of GnRH were reported. Among these, two fa milies, each containing two to four members were identified that had very h igh antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K-i < 0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) an d (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive co nformations of GnRH antagonists, yet these biocompatible constraints did no t sufficiently restrain the spatial location of the N-terminal tripeptide w ith respect to the C-terminal heptapeptide, due largely to the rotational f reedom about the bonds connecting these regions. Examination of models deri ved from NMR studies of cyclo(4-10) analogues suggested a large number of p ossible cyclic constraints such as cycle (0-8), (1-8), or (2-8). All analog ues tested with these substitutions were inactive as antiovulatory agents a t 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridgi ng positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K-i = 13 nM) or [Orn (8)] (13, K-i = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2) ,DXaa(3),Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 mu g/rat. Analogue 14 (K-i = 2.3 nM), with a [DAsp(3), Ly s(8)] bridge, was fully active at 50 mu g/rat. Loss of potency (> 20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K -i = 23 nM), Dicyclic analogues possessing the (4-10) cycle and selected (1 -6), (2-6), and (2-8) cycles led to analogues that were inactive at doses o f 500 mu g/rat or larger. Two analogues with (1-8/4-10) cycles (16, K-i = 1 .1 nM) or (3-8/4-10) cycles (22, K-i = 17 nM) showed full antiovulatory pot ency at 250 mu g/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 mu g/rat or less and K-i < 0.5 nM ) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1,1'-5/4-10) analogues met this criterion: dicyclo(1,1'-5/4-10)[A c-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5), DNal(6),Dpr(10)]GnRH (32, K-i = 0.22 nM), dicyclo(1,1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu( 5), DNal(6),Dpr(10)]GnRH (34, K-i = 0.38 nM), dicyclo(1,1'-5/4 - 10)[Ac-Asp (1)(beta Ala),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6),Dpr(10)]GnRH (40, K-i = 0.15 nM), and dicyclo(1,1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4) ,Dbu(5),DNal(6),Dpr(10)]GnRH (41, K-i = 0.24 nM). Since they differed sligh tly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive confo rmations that satisfy a very discriminating receptor, since many other clos ely related analogues were significantly less potent.