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Degarelix (FE200486, Ac-d-2Nal¹-d-4Cpa²-d-3Pal³-Ser⁴-4Aph(l-Hor)⁵-d-4Aph(Cbm)⁶-Leu⁷-ILys⁸-Pro⁹-d-Ala¹⁰-NH2) is a potent and very long acting antagonist of gonadotropin-releasing hormone (GnRH) after subcutaneous administration in mammals including humans. Analogues of degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. The duration of action was also determined in the castrated male rat assay to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release. Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and N^α-methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptor's cavity and to map the steric and ionic boundaries. Some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitutions at positions 3 ([N^β-(2-pyridyl-methyl)d-Dap³]degarelix, IC50 = 2.71 nM) (5), 7 ([Pra⁷]degarelix, IC50 = 2.11 nM) (16), and 8 ([N^δ-(IGly)Orn⁸]degarelix, IC50 = 1.38 nM) (20) and N-methylation ([N^α-methyl-Leu⁷]degarelix, IC50 = 1.47 nM) (32) yielded analogues that were equipotent to degarelix (2) in vitro (IC50 = 1.64 nM) but shorter acting in vivo. Out of the 33 novel analogues tested for the duration of action in this series, two analogues ([N^ε-cyclohexyl-Lys⁸]degarelix, IC50 = 1.50 nM) (23) and ([N^β-(IβAla)Dap⁸]degarelix, IC50 = 1.98 nM) (26) had antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%) release for >72 h after sc injection to castrated male rats at a standard dose of 50 μg/rat in 5% mannitol}. Under similar conditions analogues ([N^γ-(IGly)Dab⁸]degarelix, IC50 = 1.56 nM) (21) and ([IOrn⁸]degarelix, IC50 = 1.72 nM) (18) had a longer duration of action {inhibited LH (>96 h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of these analogues, a potential measure of their ability to be formulated for sustained release, was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer retention times. No correlation was found between retention times and antagonist potency or duration of action.

We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF1 and CRF2 receptors. Predictive methods, physicochemical measurements, and structure−activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin {cyclo(30−33)[DPhe¹²,Nle²¹,Glu³⁰,Lys³³,Nle³⁸]hCRF(12-41)} assume an α-helical conformation when interacting with their receptors. We had shown that α-helical CRF(9-41) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion¹ and later for CRF2.² More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30−33³ that would confer high affinity for both CRF1 and CRF2^2,4 in agonists and antagonists of all members of the CRF family.³ On the other hand, the substitutions that conferred ca. 100-fold CRF2 selectivity to the antagonist antisauvagine-30 {[DPhe¹¹,His¹²]sauvagine(11-40)} did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu³²-Lys³⁵ side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu³¹-Lys³⁴ side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF2. Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31−34)[DPhe¹¹,His¹²,CαMeLeu^13,39,Nle¹⁷,Glu³¹,Lys³⁴]Ac-sauvagine(8-40) (astressin2-B) with CRF2 selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF2 and CRF1 in order to confirm that astressin2-B could indeed bind to established CRF2 but not CRF1 receptor-expressing tissues. Extended duration of action of astressin2-B vs that of antisauvagine-30 is demonstrated in the CRF2-mediated animal model whereby the inhibition of gastric emptying of a solid meal in mice by urocortin administered intraperitoneally at time zero is antagonized by the administration of astressin2-B but not by antisauvagine-30 at times −3 and −6 h while both peptides are effective when given 10 min before urocortin.

A family of analogues of des-AA^1,2,5-[dTrp⁸/d2Nal⁸]-SRIF that contain a 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 was identified that has high affinity and selectivity for human somatostatin receptor subtype 1 (sst1). The binding affinities of des-AA^1,2,5-[dTrp⁸,IAmp⁹]-SRIF (c[H-Cys-Lys-Phe-Phe-dTrp-IAmp-Thr-Phe-Thr-Ser-Cys-OH], CH-275) (7), des-AA^1,5-[Tyr²,dTrp⁸,IAmp⁹]-SRIF (CH-288) (16), des-AA^1,2,5-[Tyr⁷,dTrp⁸,IAmp⁹]-SRIF (23), and des-AA^1,2,5-[dTrp⁸,IAmp⁹,Tyr¹¹]-SRIF (25) are about ¹/7, ¹/4, ¹/125, and ¹/4 that of SRIF-28 (1) to sst1, respectively, about ¹/65, ¹/130, <¹/1000, and <¹/150 that of 1 to sst3, respectively, and about or less than ¹/1000 that of 1 to the other three human SRIF receptor subtypes. A substitution of dTrp⁸ by d2Nal⁸ in 7 to yield des-AA^1,2,5-[d2Nal⁸,IAmp⁹]-SRIF (13) and in 16 to yield des-AA^1,5-[Tyr²,d2Nal⁸,IAmp⁹]-SRIF (17) was intended to increase chemical stability, selectivity, and affinity and resulted in two analogues that were less potent or equipotent with similar selectivity, respectively. Carbamoylation of the N-terminus as in des-AA^1,2,5-[dTrp⁸,IAmp⁹,Tyr¹¹]-Cbm-SRIF (27) increased affinity slightly as well as improved selectivity. Monoiodination of 25 to yield 26 and of 27 to yield 28 resulted in an additional 4-fold increase in affinity at sst1. Desamination of the N-terminus of 17 to yield 18, on the other hand, resulted in significant loss of affinity. Attempts at reducing the size of the ring with maintenance of selectivity failed in that des-AA^1,4,5,13-[Tyr²,dTrp⁸,IAmp⁹]-SRIF (33) and des-AA^{1,4,5,6,12,13}-[Tyr²,dTrp⁸,IAmp⁹]-SRIF (34) progressively lost affinity for all receptors. Both des-AA^1,2,5-[dTrp⁸,IAmp⁹,Tyr¹¹]-Cbm-SRIF (27) and des-AA^1,2,5-[dCys³,dTrp⁸,IAmp⁹,Tyr¹¹]-Cbm-SRIF (29) show agonistic activity in a cAMP assay; therefore, the structural basis for the agonist property of this family of analogues is not contingent upon the chirality of the Cys residue at position 3 as shown to be the case in 18-membered ring SRIF octapeptides. None of the high affinity structures described here showed receptor antagonism. We have prepared the radiolabeled des-AA^1,2,5-[dTrp⁸,IAmp⁹,¹²⁵ITyr¹¹]-SRIF (¹²⁵I-25) and des-AA^1,2,5-[dTrp⁸,IAmp⁹, ¹²⁵ITyr¹¹]-Cbm-SRIF (¹²⁵I-27), used them as in vitro tracers, and found them to be superior to des-AA^1,5-[¹²⁵ITyr²,dTrp⁸,IAmp⁹]-SRIF (¹²⁵I-16) for the detection of sst1 tumors in receptor autoradiography studies.

In three earlier publications (Miranda et al. J. Med. Chem. 1994, 37, 1450−1459; 1997, 40, 3651−3658; Gulyas et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575−10579) we have hypothesized that covalent constraints such as side-chain-to-side-chain lactam rings would stabilize an α-helical conformation shown to be important for the recognition and binding of the CRF C-terminus 30 residues, to CRF receptors. These studies led to the discovery of useful CRF antagonists such as α-helical CRF (α-hel-CRF) and Astressin both in vitro and in vivo. To test the hypothesis that such lactam rings may also be modulating activation of the receptor when introduced at the N-terminus of CRF, we studied the influence of the successive introduction from residues 4 to 14 of a cyclo(i,i+3)[Lysⁱ−Glu⁽ⁱ⁺³⁾] and a cyclo(i,i+3)[Gluⁱ−Lys⁽ⁱ⁺³⁾] bridge on the in vitro potency of the agonist [Ac-Pro⁴,dPhe¹²,Nle^21,38]hCRF(4-41) and related compounds. We have also introduced the favored cyclo(Glu³⁰−Lys³³) substitution found to be remarkable in several families of antagonists (such as Astressin) and in a number of CRF agonists and investigated the role of residues 4−8 on receptor activation using successive deletions. Earlier studies had shown that in both oCRF and α-helical CRF, deletion of residues 1−6, 1−7, and 1−8 led to gradual loss of intrinsic activity (IA) (from 50% IA to <10% IA) resulting in α-hel-CRF being a potent competitive antagonist. We show that acetylation of the N-terminus of these fragments generally increases potency by a factor of 2−3 with no influence on IA. While cyclo(30−33)[Ac-Leu⁸,dPhe¹²,Nle²¹,Glu³⁰,Lys³³,Nle³⁸]hCRF(8-41) (30) is the shortest reported analogue of CRF to be equipotent to CRF (70% IA), the corresponding linear analogue (31) is 120 times less potent (59% IA). Addition of one amino acid at the N-terminus {cyclo(30−33)[Ac-Ser⁷,dPhe¹²,Nle²¹,Glu³⁰,Lys³³,Nle³⁸]hCRF(7-41) (28)} results in a 5-fold increase in agonist potency and full intrinsic activity (113%). The most favored modifications were also introduced in other members of the CRF family including sauvagine (Sau), urotensin (Utn), urocortin (Ucn), and α-hel-CRF. Parallel and consistent results were obtained suggesting that the lactam cyclization at residues 29−32 and 30−33 (for the members of the CRF family with 40 and 41 amino acid residues, respectively) will induce (in the shortened agonists) a structural constraint (α-helix) that stabilizes a bioactive conformation similar to that shown in the Astressin family of CRF antagonists and that residue 8 (leucine or isoleucine) bears the sole responsibility for activation of the receptor since deletion of that residue leads to potent antagonists (Gulyas et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575−10579).

Biological potencies of three 29 amino acid growth hormone-releasing hormone analogs (GHRH[1-29]) were determined in the bovine and compared to synthetic human GHRH (44 amino acids; hGHRH[1-44]NH2) for their ability to increase serum growth hormone (GH) concentrations. Four prepubertal Holstein heifers (179 ± 10 kg) received hGHRH(1-44)NH2or analogs (D-Ala2, Nle27, Agm29GHRH[1-29], [JG-73]; D-N-MeAla2, Nle27, Agm29GHRH[1-29], [JG-75]; and desamino-Tyr1, D-Ala2, Nle27, Agm29GHRH[1-29], [JG-77]) at the following doses: 0, 6.25, 25, 100 and 400 µg/animal. All treatment-dose combinations were administered to each heifer with at least a 1-d interval between treatments. Sixteen blood samples were collected via jugular cannulas 20 min before and up to 6 h after treatment injection. There was a linear dose-dependent GH release in response to hGHRH(1-44)NH2and the three analogs. Growth hormone peak amplitudes for the three analogs were similar to those observed after administration of the hGHRH(1-44)NH2(P> .05). However, when total area under the GH response curves for each treatment was averaged over all the doses, JG-73 stimulated greater GH release than hGHRH(1-44)NH2(P< .05). Heifers injected with the 400-µg dose of hGHRH(1-44)NH2or the three analogs showed a primary release of GH followed by a secondary release 1 h later. At all other doses, only a primary GH release was observed. In conclusion, JG-73, a GHRH analog, was found to be 4.65 times more potent on a weight basis and 3.3 times more potent on a molar basis, whereas JG-75 and JG-77 were as potent as synthetic hGHRH(1-44)NH2in stimulating the release of GH in heifers.

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an α-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20−23)[dPhe¹²,Glu²⁰,Lys²³,Nle^21,38]hCRF(12-41) and of astressin {cyclo(30−33)[dPhe¹²,Nle^21,38,Glu³⁰,Lys³³]hCRF(12-41)}, two potent CRF antagonists, and of cyclo(30−33)[Ac-Leu⁸,dPhe¹²,Nle²¹,Glu³⁰,Lys³³,Nle³⁸]hCRF(8-41), the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. 1998, 41, 2614−2620 and references therein). To test the hypothesis that the G

lu²⁰−Ly

lu³⁰−Ly

In earlier reports we identified specific point substitutions (dPhe¹²,Nle^21,38), cyclization strategies [in particular, introduction of lactam rings such as that of cyclo(Glu³⁰,Lys³³)], and deletions (residues 1−7) in the CRF molecule that led to agonists. We also noted that further deletions (residues 8−14) produced antagonists such as astressin {cyclo(30−33)[dPhe¹²,Nle^21,38,Glu³⁰, Lys³³]hCRF(12-41)} (1). We hypothesized that the lactam ring promoted conformational stability to yield analogues with increased potency both in vitro and in vivo as compared to that of their linear counterparts. Additionally, we reported that cyclo(30−33)[dPhe¹²,Nle^21,38, Glu³⁰,dHis³²,Lys³³]hCRF(12-41) (3) and dicyclo(26−36,30−33)[Ac−Asp⁹,dPhe¹²,Nle^21,38,Cys²⁶, Glu³⁰,Lys³³,Cys³⁶]hCRF(9-41) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30−33 (previous paper:  Koerber et al. J. Med. Chem. 1998, 41). To increase the potency of 1 and/or 3 in vivo, we extended their chain length by one (5−8), two (9, 10), and three (11, 12) residues at the N-terminus and acetylated (6, 8, 10, 12). Of the compounds tested for duration of action (1, 3−6, 8), we found 6 and 8 to be slightly longer-acting than astressin or [dHis³²]astressin, while their potencies in vitro were not significantly different from that of 3. Additionally, we introduced CαMe-leucine residues in lieu of leucine at positions 14, 15, 19, 27, and 37 in [dHis³²]astressin. The analogue [CαMe-Leu²⁷,dHis³²]astressin (16) was more potent (although not statistically in all cases) than the other four analogues in vitro. While acetylation of the N-terminus of 16 (i.e., 18) or of [CαMe-Leu²⁷]astressin (i.e., 19) did not have a significant effect on in vitro potency, elongation of the N-terminus by one or three residues in addition to acetylation resulted in cyclo(30−33)[dPhe¹²,Nle²¹,CαMe-Leu²⁷,Glu³⁰,dHis³²,Lys³³,Nle³⁸]Ac-hCRF(11-41) (21), cyclo(30−33)[dPhe¹²,Nle²¹,CαMe-Leu²⁷,Glu³⁰,Lys³³,Nle³⁸]Ac-hCRF(9-41) (22), and cyclo(30−33)[dPhe¹², Nle²¹,CαMe-Leu²⁷,Glu³⁰,dHis³²,Lys³³,Nle³⁸]Ac-hCRF(9-41) (23) that were longer-acting than 6 and 8 (ca. 2 h inhibition of ACTH secretion at 25 μg/adrenalectomized rat). Analogues 22 and 23 were also more potent than astressin at reversing intracisternal CRF- and abdominal surgery-induced delay of gastric emptying in conscious rats.

Hypothesis driven and systematic structure−activity relationship (SAR) investigations have resulted in the development of effective central nervous system (CNS) antagonists of corticotropin (ACTH)-releasing factor (CRF) such as α-helical CRF(9-41)³ and analogues of our assay standard [DPhe¹²,Nle^21,38]hCRF(12-41).⁴ On the other hand, equally potent CRF antagonists that block the hypothalamic/pituitary/adrenal (HPA) axis had not been described until recently.⁵ Predictive methods, physicochemical measurements (nuclear magnetic resonance spectrometry and circular dichroism spectroscopy), and SAR studies suggest that CRF and its family members (urotensins and sauvagine) assume an α-helical conformation when interacting with CRF receptors.^6,7 To further test this hypothesis, we have systematically scanned the hCRF(9-41) or hCRF(12-41) sequences with an i−(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown could maintain or enhance α-helical structure. From this series we have identified seven analogues that are either equipotent to, or 3 times more potent than, the assay standard; in addition, as presented earlier⁵ cyclo(30−33)[DPhe¹²,Nle^21,38,Glu³⁰,Lys³³]hCRF(12-41) (astressin) is 32 times more potent than the assay standard in blocking ACTH secretion in vitro (rat pituitary cell culture assay). In vivo, astressin is also significantly more potent than earlier antagonists at reducing hypophysial ACTH secretion in intact stressed or adrenalectomized rats.⁵ Since the corresponding linear analogues that were tested are significantly less potent, our interpretation of the increased potency of the cyclic analogues is that the introduction of the side chain to side chain bridging element (Glu³⁰---Lys³³, and to a lesser extent that of Glu¹⁴---Lys¹⁷, Glu²⁰---Lys²³, Glu²³---Lys²⁶, Glu²⁶---Lys²⁹, Glu²⁸---Lys³¹, Glu²⁹---Lys³², and Glu³³---Lys³⁶) induces and stabilizes in the receptor environment a putative α-helical bioactive conformation of the fragment that is not otherwise heavily represented. The effect of the introduction of two favored substitutions [(cyclo(20−23) and cyclo(30−33)] yielded 37 with a potency 8 times that of the assay standard but actually 12 times less than expected if the effect of the two cycles had been multiplicative. These results suggest that the pituitary CRF receptor can discriminate between slightly different identifiable conformations, dramatically illustrating the role that secondary and tertiary structures play in modulating biological signaling through specific protein−ligand interactions.

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = gamma-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Phthaloyl‐L‐glutamic acid (I) is converted to the anhydride (II) which is coupled with the aminosulfonic acid (III) in the presence of tetramethylguanidine (IV).