Design of Monocyclic (1−3) and Dicyclic (1−3/4−10) Gonadotropin Releasing Hormone (GnRH) Antagonists

Careful analysis of the NMR structures of cyclo(4−10)[Ac-Δ3Pro,dFpa,dTrp,Asp,dNal6,Dpr10]GnRH, dicyclo(4−10/5−8)[Ac-dNal1,dCpa2,dTrp3, Asp4,Glu,dArg,Lys8,Dpr10]GnRH, and dicyclo(4−10/5,5‘−8)[Ac-dNal1,dCpa2,dPal3,Asp4, Glu5(Gly),dArg6,Dbu8,Dpr10]GnRH showed that, in the N-terminal tripeptide, a type II β-turn around residues 1 and 2 was probable along with a γ-turn around dTrp3/dPal3. This suggested the possibility of constraining the N-terminus by the introduction of a cyclo(1−3) scaffold. Optimization of ring size and composition led to the discovery of cyclo(1−3)[Ac-dAsp1,dCpa2,dLys3,dNal6,dAla10]GnRH (5, K i = 0.82 nM), cyclo(1,1‘−3)[Ac-dAsp1(Gly),dCpa2,dOrn3,dNal6,dAla10]GnRH (13, K i = 0.34 nM), cyclo(1,1‘−3)[Ac-dAsp1(Gly),dCpa2,dLys3,dNal6,dAla10]GnRH (20, K i = 0.14 nM), and cyclo(1,1‘−3)[Ac-dAsp1(βAla), dCpa2,dOrn3,dNal6,dAla10]GnRH (21, K i = 0.17 nM), which inhibited ovulation significantly at doses equal to or lower than 25 μg/rat. These results were particularly unexpected in view of the critical role(s) originally ascribed to the side chains of residues 1 and 3. Other closely related analogues, such as those where the [dAsp1(βAla), dOrn3] cycle of 21 was changed to [dOrn1(βAla), dAsp3] of cyclo(1,1‘−3)[Ac-dOrn1(βAla),dCpa2,dAsp3,dNal6,dAla10]GnRH (22, K i = 2.2 nM) or where the size of the cycle was conserved and [dAsp1(βAla), dOrn3] was replaced by [dGlu1(Gly), dOrn3] as in cyclo(1,1‘−3)[Ac-dGlu1(Gly),dCpa2,dOrn3,dNal6,dAla10]GnRH (23, K i = 4.2 nM), were approximately 100 and 25 times less potent in vivo, respectively. Analogues with ring sizes of 18 {cyclo(1,1‘−3)[Ac-dGlu1(Gly),dCpa2,dLys3,dNal6,dAla10]GnRH (24)} and 19 {cyclo(1,1‘−3)[Ac-dGlu1(βAla),dCpa2,dLys3,dNal6,dAla10]GnRH (25)} atoms were also less potent than 21 with slightly higher K i values (1.5 and 2.2 nM, respectively). These results suggested that the N-terminal tripeptide was likely to assume a folded conformation favoring the close proximity of the side chains of residues 1 and 3. The dicyclic analogue dicyclo(1−3/4−10)[Ac-dAsp1,dCpa2,dLys3,Asp4,dNal6,Dpr10]GnRH (26) was fully active at 500 μg, with a K i value of 1 nM. The in vivo potency of 26 was at least 10-fold less than that of monocyclic cyclo(1−3)[Ac-dAsp1,dCpa2,dLys3,dNal6,dAla10]GnRH (5); this suggested the existence of unfavorable interactions between the now optimized and constrained (1−3) and (4−10) cyclic moieties that must interact as originally hypothesized. Tricyclo(1−3/4−10/5−8)[Ac-dGlu1,dCpa2, dLys3,Asp4,G