PubMed2000
With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was systematically explored. In an earlier publication, cyclo[Asp(4)-Dpr(10)]GnRH antagonists with high potencies in vitro and in vivo had been identified.(1) Independently from Dutta et al. (2) and based on structural considerations, the cyclic [Glu(5)-Lys(8)] constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4-10) and (5-8) and dicyclic (4-10/5-8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Asp(4),DArg(6),Xaa(10)]GnRH with Xaa = D/LAgl (1, K(i) = 1.3 nM) or Dpr (2, K(i) = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 microgram at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K(i) = 10 nM) or cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Glu(4),DArg(6),D/LAgl(10)]GnRH (4, K(i) = 1.3 nM). Cyclo(5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Glu(5),DArg++ +(6),Lys(8), DAla(10)]GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K(i) = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5-8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5'-8)[Ac-DNal(1), DCpa(2),DPal(3),Glu(5)(betaAla),DArg(6),(D or L)Agl,(8)DAla(10)]GnRH 14 (K(i) = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 microgram/rat). Dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Asp (4),Glu(5),DArg(6), Lys(8),Dpr(10)]GnRH (24, K(i) = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4-10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5-8) bridge was varied. For example, the substitution of Xbb(5') by Gly (30, K(i) = 0.16 nM), Sar (31, K(i) = 0.20 nM), Phe (32, K(i) = 0.23 nM), DPhe (33, K(i) = 120 nM), Arg (36, K(i) = 0.20 nM), Nal (37, K(i) = 4.2 nM), His (38, K(i) = 0.10 nM), and Cpa (39, K(i) = 0.23 nM) in cyclo(4-10/5,5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4),G lu(5)(Xbb(5')), DArg(6),Dbu,(8)Dpr(10)]GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K(i) = 2.4 nM; and 8, K(i) = 450 nM), cyclo[Glu(5)-Aph(8)] (16, K(i) = 20 nM; and 17, K(i) = 0.28 nM), or cyclo[Asp(5)-/Glu(5)-/Asp(5)(Gly(5'))-Amp(8)] (19, K(i) = 1.3 nM; 22, K(i) = 3.3 nM; and 23, K(i) = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological activity of substituting DCpa or DFpa at position 2, DPal or DTrp at position 3, and DArg, DNal, or DCit at position 6 are also discussed. Interestingly, monocyclo(5-8)[Glu(5), DNal(6),Lys(8)]GnRH (18, K(i) = 1. (ABSTRACT TRUNCATED)