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Careful analysis of the NMR structures of cyclo(4-10)[Ac-Delta(3)Pro(1),DFpa(2),DTrp(3),Asp(4),DNal(6),Dpr(10)]-GnRH, dicyclo(4- 10/5 - 8)[Ac-DNal(1),DCpa(2),DTrp(3), Asp(4),Glu(5),DArg(6),Lys(8),Dpr(10)]GnRH, and dicyclo(4-10/5,5’-8)[Ac-DNa1,DCpa(2),DPal(3),Asp(4), Glu(5)(Gly),DArg(6),Dbu(8),Dpr(10)]GnRH showed that, in the N-terminal tripeptide, a type II beta-turn around residues 1 and 2 was probable along with a gamma-turn around DTrp(3)/DPal(3). 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-DAsp(1),DCpa(2),DLys(3),DNal(6),DAla(10)]GnRH (5, K-i = 0.82 nM), cyclo(1,1-’3)-[Ac-DAsp(1)(Gly),DCpa(2),DOrn(3),DNal(6),DAla(10)]GnRH(13, K-i = 0.34 nM), cyclo(1, 1’-3)[Ac-DAsp(1)(beta Ala),DCpa(2),DOrn(3),DNal(6),DAla(10)]GnRH (20, K-i = 0.14 nM), and cyclo(1,1’-3)[Ac-DAsp(1)(beta Ala), DCpa(2),DOrn(3),DNal(6),DAla(10)]GnRH (21, K-i = 0.17 nM), which inhibited ovulation significantly at doses equal to or lower than 25 mu g/rat. These results were particularly unexpected in view of the critical role(s) originally ascribed to the side chains of residues 1 and 3.(1) Other closely related analogues, such as those where the [DAsp(1)(beta Ala), DOrn(3)] cycle of 21 was changed to [DOrn(1)(beta Ala), DAsp(3)] of cyclo(1,1’-3)[Ac-DOrn(1)(beta Ala),DCpa(2),DAsp(3),DNal(6),DAla(10)]GnRH (22, K-i = 2.2 nM) or where the size of the cycle was conserved and [DAsp(1)(beta Ala), DOrn(3)] was replaced by [DGlu(1)(Gly), DOrn(3)] as in cyclo(1,1’-3)[Ac-DGlu(1)(Gly),DCpa(2),DOrn(3),DNal(6),DAla(10)] 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-DGlu(1)(Gly),DCpa(2),DLys(3),DNal(6),DAla(10)]GnRH (24)} arid 19 {cyclo(1,1’-3)[Ac-DGlu(1)(beta Ala),DCpa(2),DLys(3),DNal(6),DAla(10)]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-DAsp(1),DCpa(2),DLys(3),Asp(4),DNal(6),Dpr(10)] GnRH (26) was fully active at 500 mu g, 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-DAsp(1),DCpa(2),DLys(3),DNal(6),DAla(10)]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-DGlu(1),DCpa(2), DLys(3),Asp(4),Glu(5),DNal(6),Lys(8),Dpr(10)] GnRH (27) was inactive at 500 mu g/rat with a corresponding low affinity (K-i = 4.6 nM) when compared to those of the most potent analogues (K-i < 0.5 nM).