Further structure-activity relationship studies of a series of substituted uracils at the 1, 3, and 5 positions resulted in the discovery of several potent antagonists of the human gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylglycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-dione (R-13b, NBI 42902) displayed subnanomolar binding affinity (K(i) = 0.56 nM) and was a potent functional antagonist (IC(50) = 3.0 nM in Ca(2+) flux assay) at the human GnRH receptor. It also bound to the monkey GnRH receptor with high affinity (K(i) = 3.9 nM). In addition, R-13bhad good plasma exposure in cynomolgus monkeys after oral administration, with a C(max) of 737 ng/mL and an AUC of 2392 ng/mL.h at a 10 mg/kg dose. Moreover, oral administration of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum levels of luteinizing hormone. These results demonstrate that compounds from this series of uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman primates.

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...

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.

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-DNal(1),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)(Gly),DCpa(2),DLys(3),DNal(6),DA la(10)]GnRH (20, K(i) = 0.14 nM), and cyclo(1,1'-3)[Ac-DAsp(1)(betaAla), 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 microgram/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)(betaAla), DOrn(3)] cycle of 21 was changed to [DOrn(1)(betaAla), DAsp(3)] of cyclo(1,1'-3)[Ac-DOrn(1)(betaAla), 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)(betaAla), 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),DA la(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),DA la(10)]GnRH (24)¿ and 19 ¿cyclo(1,1'-3)[Ac-DGlu(1)(betaAla),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 microgram, 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 microgram/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).