Iterative approach to the discovery of novel degarelix analogues: substitutions at positions 3, 7, and 8. Part II

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 in order 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, 8 and Nα-methylation at positions 6, 7 and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptors 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 like bioavailability, solubility, intra- or inter-molecular hydrogen bond forming capacity and ability to bind carrier proteins. Substitutions at positions 3 ([Nβ-(2-pyridyl-methyl)d-Dap3]degarelix, IC50 = 2.71 nM) (5), 7 ([Pra7]degarelix, IC50 = 2.11 nM) (16), 8 ([Nδ-(IGly)Orn8]degarelix, IC50 = 1.38 nM) (20), and N-methylation ([Nα-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 ([Nε-cyclohexyl-Lys8]degarelix, IC50 = 1.50 nM) (23) and ([Nβ-(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 ([Nγ-(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, antagonist potency or duration of action.

antagonist, azaline B, degarelix, endometriosis, GnRH receptor, gonadotropin-releasing hormone, luteinizing hormone, prostate cancer

Gonadotropin-releasing hormone (GnRH) antagonists are used in the protocols for ovulation induction and are recognized as potential drugs for the management of sex steroid-dependent pathophysiologies, such as hormone-responsive prostate cancers and, in females, the management or treatment of breast and gynecological cancers, endometriosis, precocious puberty, uterine myoma, ovarian hyperandrogenism and premenstrual syndrome.1–4 Most of these disorders can be treated with long acting preparations of the super agonists, which desensitize the gonadotrophs after approximately two weeks of treatment. An optimized antagonist will likely become the preferred choice over the agonist in the clinic because it avoids the initial up-regulation of the gonadotropin-gonadal axis, leads to rapid and predictable recovery, achieves more profound inhibition of gonadotropins and can be used as a diagnostic test of gonadotropin-dependent gonadal dysfunction. The present study of the effect of substitutions at position 3 of degarelix is an extension of an earlier study5 which demonstrated that the non aromatic substitution of d3Pal by d-glutamine in Ac-d-2Nal1-d-4Cpa2-d-3Pal3-Ser4-4Aph(l-Hor)5-d-4Aph(Ac)6-Leu7-Ilys8-Pro9-d-Ala10-NH2 was compatible with high potency as well as very long duration of action (i.e. > 80% inhibition of LH in the castrated male rat after sc administration of 50 µg in 5% mannitol for > 96 h) whereas the equivalent d-asparagine-containing analogue was comparatively shorter acting. This suggested that very minor substitutions at position 3 might possibly have an unexpected impact on duration of action with minimal effect on pA2 (the × intercept in a Schild analysis of receptor antagonism, the concentration of antagonist in log terms which right-shifts agonist dose response curves two fold). Substitutions at position 7 explore the effect of the introduction of functional groups such as a primary amide (in asparagine), imidazole (in histidine), triple bonds (in propargylglycine), phenol (in tyrosine) and sulfhydryl (in cysteine) on the antagonist potency and the duration of action in inhibition of luteinizing hormone (LH) release. Early studies6 suggested that a hydrophobic moiety would be preferable, yet the parent analogues were structurally quite different from degarelix and the question remained whether side chains prone to hydrogen bonding could be tolerated in degarelix for intramolecular stabilization. Additionally, we investigated the effect of novel substitutions at position 8 of degarelix. It is well documented that substitution of arginine by isopropyl-lysine at position 8 in GnRH antagonists yields potent analogues in antiovulatory and GnRH receptor assays with low potency to release histamine.7 It was therefore hypothesized that other closely related structures to that of ILys might be even more favorable. Most substitutions are based on acylation of the side chains of dibasic residues of α,β-diaminopropionic acid (Dap) and α,γ-diaminobutyric acid (Dab) as well as ornithine reminiscent to our work on betidamino acids (acylated α,α-diaminoacetic acids or amino-glycines).8 Finally, the key residues encompassing a β-turn9 at positions 6, 7 and 8 of degarelix were Nα-methylated to confer enzymatic stability,10 increasing solubility11 and to induce or disrupt sterical constraints with possible effects on activities.

and Discussion Synthesis, Purification, and Chemical Characterization (Table 1) Table 1. | No | Compound | Purity | tRc (min) | MSd (M + H)+ | pIC50e | IC50f (nM) | Duration of actiong | || |---|---|---|---|---|---|---|---|---|---| | HPLCa | CZEb | Calc. | Obs. | Avg. ± SEM | ||||| | 1 | [Ac-d-2Nal1,d-4Cpa2,d-3Pal3,4Aph(Atz)5,d-4Aph(Atz)6,Ilys8,d-Ala10] GnRH azaline B | 99 | 99 | 27.4 | 1612.8 | 1612.7 | 8.9 ± 0.07 | 1.4 | Long | | 2 | [Ac-d-2Nal1,d-4Cpa2,d-3Pal3,4Aph(l-Hor)5,d-4Aph(Cbm)6,Ilys8,d-Ala10] GnRH degarelix | 96 | 98 | 27.9 | 1631.8 | 1631.9 | 8.8 ± 0.03 | 1.64 | Very long | | 3 | [Nβ, Nβ-di-(2-pyridyl-methyl)d-Dap3]degarelix | 97 | 99 | 33.4 | 1751.8 | 1751.6 | 8.3 ± 0.01 | 5.31 | Short | | 4 | [Nγ, Nγ-di-(2-pyridyl-methyl)d-Dab3]degarelix | 89 | 93 | 33.5 | 1765.8 | 1765.8 | 7.6 ± 0.01 | 22.58 | Short | | 5 | [Nβ-(2-pyridyl-methyl)d-Dap3]degarelix | 95 | 98 | 29.5 | 1660.8 | 1660.8 | 8.6 ± 0.05 | 2.71 | Short | | 6 | [Nγ-(2-pyridyl-methyl)d-Dab3]degarelix | 95 | 97 | 28.8 | 1674.8 | 1674.8 | 8.4 ± 0.18 | 3.79 | Short | | 7 | [Nβ-(2-quinolyl-methyl)d-Dap3]degarelix | 94 | 96 | 35.1 | 1710.8 | 1710.6 | 8.4 ± 0.07 | 4.25 | Short | | 8 | [Nγ-(2-quinolyl-methyl)d-Dab3]degarelix | 95 | 95 | 34.8 | 1724.8 | 1724.6 | 8.3 ± 0.02 | 5.52 | Short | | 9 | [Nβ-(2-imidazolyl-methyl)d-Dap3]degarelix | 95 | 96 | 27.6 | 1649.8 | 1649.6 | 8.5 ± 0.06 | 3.50 | Intermediate | | 10 | [Nγ-(2-imidazolyl-methyl)d-Dab3]degarelix | 98 | 97 | 26.7 | 1663.8 | 1663.8 | 7.9 ± 0.03 | 13.47 | Short | | 11 | [Asn7]degarelix | 97 | 97 | 22.5 | 1632.7 | 1632.6 | 8.0 ± 0.03 | 9.18 | Short | | 12 | [Nγ-methyl-Asn7]degarelix | 97 | 98 | 23.2 | 1646.7 | 1646.8 | 7.6 ± 0.01 | 25.79 | Short | | 13 | [Nγ, Nγ-dimethyl-Asn7]degarelix | 98 | 98 | 24.2 | 1660.7 | 1660.7 | 7.3 ± 0.08 | 48.91 | Short | | 14 | [His7]degarelix | 95 | 99 | 22.7 | 1655.7 | 1655.7 | 8.0 ± 0.10 | 10.64 | Short | | 15 | [Tyr7]degarelix | 95 | 99 | 25.1 | 1681.7 | 1681.7 | 8.5 ± 0.01 | 2.85 | Intermediate | | 16 | [Pra7]degarelix | 96 | 97 | 24.9 | 1613.7 | 1613.7 | 8.7 ± 0.03 | 2.11 | Short | | 17 | [Cys7]degarelix | 97 | 96 | 25.3 | 1621.7 | 1621.7 | 6.7 ± 0.01 | 178.19 | Short | | 18 | [IOrn8]degarelix | 99 | 99 | 28.1 | 1617.7 | 1617.7 | 8.8 ± 0.14 | 1.72 | Long* | | 19 | [Nε-(IGly)Lys8]degarelix | 99 | 98 | 30.6 | 1688.8 | 1688.7 | 8.7 ± 0.11 | 1.83 | Short | | 20 | [Nδ-(IGly)Orn8]degarelix | 98 | 99 | 30.1 | 1674.8 | 1674.7 | 8.9 ± 0.12 | 1.38 | Intermediate | | 21 | [Nγ-(IGly)Dab8]degarelix | 97 | 98 | 30.3 | 1660.7 | 1660.7 | 8.8 ± 0.09 | 1.56 | Long* | | 22 | [Nβ-(IGly)Dap8]degarelix | 95 | 98 | 30.6 | 1646.7 | 1646.8 | 8.8 ± 0.02 | 1.54 | Intermediate | | 23 | [Nε-cyclohexyl-Lys8]degarelix | 98 | 97 | 31.7 | 1671.8 | 1671.7 | 8.8 ± 0.08 | 1.50 | Long | | 24 | [Nγ-(Nα-cyclohexyl-Gly)Dab8]degarelix | 98 | 98 | 35.8 | 1700.8 | 1700.7 | 8.8 ± 0.15 | 1.77 | Short | | 25 | [Nβ-(Nα-cyclohexyl-Gly)Dap8]degarelix | 99 | 97 | 36.0 | 1686.8 | 1686.7 | 8.7 ± 0.14 | 2.01 | Short | | 26 | [Nβ-(IβAla)Dap8]degarelix | 96 | 98 | 28.3 | 1660.7 | 1660.7 | 8.7 ± 0.25 | 1.98 | Long | | 27 | [Nβ-(IβAla)-Nβ-methyl-Dap8]degarelix | 98 | 98 | 29.0 | 1674.8 | 1674.7 | 8.7 ± 0.19 | 1.97 | Intermediate | | 28 | [Nβ-(ICbm)Dap8]degarelix | 98 | 97 | 31.7 | 1632.7 | 1632.7 | 8.8 ± 0.16 | 1.62 | Short | | 29 | [Nγ-(ICbm)Dab8]degarelix | 99 | 98 | 31.8 | 1646.7 | 1646.7 | 8.6 ± 0.06 | 2.24 | Short | | 30 | [Nδ-(l-Hor)Orn8]degarelix | 98 | 98 | 27.2 | 1715.7 | 1715.7 | 8.8 ± 0.16 | 1.77 | Short | | 31 | [Nα-methyl-d-4Aph(Cbm)6]degarelix | 97 | 98 | 30.2 | 1645.8 | 1645.6 | 8.7 ± 0.15 | 1.85 | Short | | 32 | [Nα-methyl-Leu7]degarelix | 98 | 99 | 30.3 | 1645.8 | 1645.7 | 8.8 ± 0.11 | 1.47 | Short | | 33 | [Nα-methyl-Ilys8]degarelix | 99 | 96 | 29.1 | 1645.8 | 1645.8 | 7.3 ± 0.01 | 52.24 | Short | Percentage purity determined by HPLC using buffer system A; TEAP, pH 2.30, buffer system B, 60% CH3CN/40% A under gradient conditions (30% to 80% B over 50 min), at flow rate of 0.2 mL/min on a Vydac C18 column (0.21 × 15 cm, 5 µm particle size, 300 Å pore size). Detection at 214 nm. Percentage purity determined by capillary zone electrophoresis (CZE) using a Beckman P/ACE System 2050 controlled by an IBM Personal system/2 model 50Z; field strength of 15 kV at 30 °C. Buffer, 100 mM sodium phosphate (85:15, H2O:CH3CN), pH 2.50, on a Agilent µSil bare fused-silica capillary (75 µm i.d. × 40 cm length). Detection at 214 nm. Retention times under gradient conditions (30% to 80% B over 50 min); buffer system A; TEAP, pH 7.0, buffer system B, 60% CH3CN/40% A. Mass spectra (MALDI-MS) were measured on an ABI-Voyager DESTR instrument using saturated solution of α-cyano-4-hydroxycinnamic acid in 0.3% trifluoroacetic acid and 50% acetonitrile as matrix. The calculated [M + H]+of the monoisotope compared with the observed [M + H]+ monoisotopic mass. The pIC50 is the negative log of the IC50 in Molar, as determined in the GnRH reporter gene assay. IC50 is the concentration of antagonist required to repress the GnRH induced response by 50% in the reporter gene assay in HEK-293 cells expressing the human GnRH receptor and a GnRH-responsive stably integrated luciferase reporter gene. Castrated male rat assay. Duration of action: very long = over 80% inhibition of LH release for more than 120 h; long* = over 80% inhibition of LH release at 96 h but not at 120 h; long = over 80% inhibition of LH release at 72 h but not at 96 h; intermediate = partial inhibition of LH release at 72 h; short = over 80% inhibition of LH release at 3 h but not at 72 h. Analogues were synthesized by the solid-phase peptide synthesis (SPPS) methodology on a p-methylbenzhydrylamine resin (MBHA-resin, tert-butyloxycarbonyl (Boc) strategy) using protocols previously described12 or shown below. In most cases, analogues were obtained by the introduction of each of the side chain substituents at positions 3, 5, 6 and 8 on the partially deprotected peptido-resin obtained after the removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group with 30% piperidine in N-methylpyrrolidinone (NMP), (for example, after the introduction of Boc-d-Dab/d-Dap(Fmoc) at position 3, Boc-d-4Aph(Fmoc) at position 5, Boc-4Aph(Fmoc) at position 6 and Boc-Dab/Dap/Orn/Lys(Fmoc) at position 8). The reference compounds azaline B (1) and degarelix (2) were synthesized using published protocols.13,14 The synthesis of all other analogues deserves further comment. In all of the analogues, the side chain substitutions at position 5 and 6 were carried out during the elongation of the peptide chain on the resin. The carbamoylation14 at position 6 was carried out by the reaction of tert-butyl isocyanate in dimethylformamide (DMF) with the free ω-amino function of d-p-aminophenylalanine (d-4Aph) on the otherwise fully protected resin-bound peptides. Coupling of l-hydroorotic acid (l-Hor) to the resin-bound deprotected ω–amino group of l-4Aph at position 5 was mediated by N, N’-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in NMP. The synthesis of analogues 3–10 involved the reductive alkylation of the deprotected side-chain amino group of d-Dap/d-Dab at position 3 on the fully assembled peptide resin, [Ac-d-2Nal-d-4Cpa-d-Dap/Dab(Fmoc)-Ser(Bzl)-4Aph(l-Hor)-d-4Aph(Cbm)-Leu-ILys(Nε-Cbz)-Pro-d-Ala]MBHA-resin. A number of reductive alkylation protocols on solid phase have been reported in the literature.15–18 Hocart et al.15,16 have described the reductive alkylation of the side chain of d-Lys with various aldehydes and ketones in the presence of sodium cyanoborohydride (NaCNBH3) at position 6 of the GnRH antagonist [Ac-d-2Nal1-d-Phe2-d-Phe3-Ser4-Tyr5-d-Lys6-Phe7-Leu8-Pro9-d-Ala10-NH2] in an attempt to reduce the histamine-releasing activity. In our hands, the reductive alkylation of the free amino group on the resin with an aromatic aldehyde in the presence of NaCNBH3 gave the mixture of mono and dialkylated products. The formation of the dialkylated product was observed even when the reaction time was shortened to 20 min. The Nβ, Nβ- or Nγ, Nγ- dialkylated peptides 3 and 4 were prepared by exhaustive reductive alkylation (2 × 3 h) of the deprotected amino group of d-Dap and d-Dab respectively, with an excess of 2-pyridinecarboxaldehyde and NaCNBH3 in DMF on the resin (see Experimental Section). In the synthesis of analogues 5–10, we used the stepwise or indirect reductive alkylation19 involving the pre-formation of the intermediate imine on the resin-bound peptide followed by reduction in a separate step. The synthesis of analogues 5–10 involved the reaction of an excess of aromatic aldehydes [2-pyridinecarboxaldehyde (in 5, 6), 2-quinolinecarboxaldehyde (in 7, 8) and 2-imidazolecarboxaldehyde (in 9, 10)] in DMF with the unprotected orthogonal amino function of amino acids (d-Dap/d-Dab) on the otherwise fully protected and assembled peptide resins resulting in aldamines. A significant reaction time (4 h) was required and in the second step, the reduction of the aldamine was carried out directly in DMF with an excess of NaCNBH3 to give the desired secondary amine. The formation of the secondary amine on the resin was qualitatively determined by the chloranil test.20 Most of the unusual amino acids substituted at positions 7 and 8 of degarelix were obtained commercially or synthesized as described in the literature. However, the synthesis of two new amino acids Nα-Boc-Lys(Nε-cyclohexyl, Nε-Cbz) and IβAla(Nα-Cbz) substituted at position 8 of degarelix deserves further comment. The Nα-Boc-Lys(Nε-cyclohexyl, Nε-Cbz) was prepared via reductive alkylation of Nα-Boc-Lys(Nε-Cbz) with cyclohexanone under Pd/C-H2 conditions, followed by secondary amine protection using Cbz-Cl. The synthesis of IβAla(Nα-Cbz) involved direct nucleophilic displacement reaction of 3-bromopropionic acid with isopropylamine to give isopropyl-βAla (IβAla) followed by Cbz protection (see Experimental Section). Analogues 11–18 and 23 were synthesized by introducing different amino acids at position 7 or 8 during the elongation of the peptide chain and using the strategy reported for the synthesis of degarelix.14 The synthesis of analogues 19–22, 24–26 and 30, was accomplished by DIC and HOBt mediated coupling reaction of the resin-bound deprotected orthogonal amino group at position 8 with Gly(Nα-isopropyl, Nα-Cbz) (in 19–22), Gly(Nα-cyclohexyl, Nα-Cbz) (in 24 and 25), βAla(Nα-isopropyl, Nα-Cbz) (in 26) and l-Hor (in 30). Further elongation of the peptide chain, HF cleavage and purification afforded the desired analogues. The synthesis of 28 and 29 involved the reaction of isopropyl isocyanate with the unprotected orthogonal amino function of Dap (in 28) or Dab (in 29) at position 8 on the otherwise fully protected and assembled peptide resins. The introduction of the Nβ-methyl group in 27 and Nα-methyl group in analogues 31–33 was achieved during the elongation of the peptide chain on the partially assembled peptide resin using the published procedure of Kaljuste et al.21 The protected peptido-resins were cleaved and deprotected in anhydrous HF (1.5 h at 0–5 °C) in the presence of a scavenger (anisole). The crude peptides were purified by reversed-phase HPLC (RP-HPLC) in two steps and isolated as their trifluoroacetate (TFA) salts. The analytical techniques used for the characterization of the analogues included RP-HPLC with two different solvent systems (acidic and neutral) and capillary zone electrophoresis (CZE). With very few exceptions, all of the analogues were greater than 98% pure. Mass spectrometric analysis supported the identity of the intended structures, Table 1. Biological characterization (Table 1, Figure 1 and Figure 2) Analogues were tested in vitro for their antagonism at the GnRH receptor in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor and a stably integrated luciferase reporter gene.14,22 The antagonism of the GnRH agonist-induced response by each analogue was determined at several concentrations and reported as IC50, the concentration required to suppress the response in the reporter gene assay by 50%, Figure 1. Since there is strong evidence showing that most of the GnRH antagonists (except for those that are very short acting) inhibit LH secretion maximally to about the same level in the highly reproducible in vivo castrated male rat assay,23,24 the most efficient analogues are those with the longest duration of action suggesting the use of this assay for screening purposes as validated by the discovery of degarelix (FE200486).14 In short, ten days after castration, rats (5 or more per group) were injected on a Friday with the excipient (50 µL of 5% mannitol), degarelix and azaline B (50 µg/50 µL 5% mannitol) as an internal standard, and the novel analogues (50 µg/50 µL 5% mannitol). Blood sampling was performed pre-dose and then at 3 h, 72 h, 96 h, 120 h, 144 h, 168 h and occasionally at 192 h post-sc administration (bloods were not collected over weekends). The effects of the test compounds on the gonadotropic axis were determined by measurement of plasma LH levels using a radioimmunoassay24 (Figure 2). By our definition, an analogue is very long acting when 50 µg in 5% mannitol in a volume of 50 µL injected sc results in an inhibition of LH (>80%) that lasts for more than 120 h. Under the same conditions, an analogue that is long* acting (see Table 1) will inhibit LH (>80%) secretion for at least 96 h; an analogue that is long acting will inhibit LH (>80%) secretion for at least 72 h; an analogue that is intermediate acting will inhibit LH secretion only partially at 72 h; and an analogue that is short acting will inhibit LH (>80%) secretion at 3 h but not at 72 h.

and Discussion The overall rationale for the synthesis of the different analogues of degarelix was presented in our introduction and consists of probing the steric boundaries, ionic properties and hydrogen bond-forming capacity of the pharmacophores at positions 3, 7 and 8, known to be critical yet not necessarily optimized in the analogues reported so far.6,25 Additionally, some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues through altered solubility, stability and ability to bind to carrier proteins. There is precedence for favorable substitutions at position 3; it includes glutamine and carbamoylation of Dap and Dab.5 Another favorable substitution at position 3 includes the aminotriazole functionality.13 Yet there is no available data on the effect of alkylation and dialkylation of a ω-NH2 at this position. To mimic the functionality of the favored d-3Pal3 found in azaline B,26 acyline,27 degarelix14 and many other analogues, we carried out reductive alkylation on d-Dap/d-Dab to generate a variety of amino acids that can provide a combined modulation of steric effects, basicity, aromaticity and hydrophobicity at position 3. The reductive alkylation with 2-pyridinecarboxaldehyde under two different experimental conditions resulted in the formation of the mono- and di-alkylated products (5, 6 and 3, 4, respectively). Comparison of the antagonist potency data indicated that increasing the length of the side chain from Dap (in 3 and 5) to Dab (in 4 and 6) resulted in ca. 2 to 4-fold loss of potency (compare IC50s of 4 = 22.58 nM versus 3 = 5.31 nM and of 6 = 3.79 nM versus 5 = 2.71 nM). All four analogues are short acting. A similar set of derivatives (7–10) was significantly less potent in vitro, likely due to increased steric hindrance (7, 8) and charge distribution (9, 10). Interestingly, 9 (with intermediate duration of action) showed some extended duration of action over 10 (with short duration of action). Several different amino acids have been introduced at position 7 of GnRH agonists and antagonists in the past.6 Interestingly, leucine, found in mammalian GnRH, is not a conserved amino acid in many other species: tryptophan is found in salmon GnRH,28 dogfish GnRH,29 chicken II GnRH,30 lamprey I GnRH31 and lamprey III GnRH,32 phenylalanine is found in tunicate GnRH-133 and tunicate GnRH-3,34 histidine is found in tunicate GnRH-2,33 and tyrosine is found in tunicate GnRH-5 and 6.34 In the present study, we have introduced amino acids such as Asn (in 11), Asn(Me) (in 12), Asn(Me)2 (in 13), Pra (in 16) or Cys (in 17) along with His (in 14) and Tyr (in 15) found in GnRHs of other species (see above). It is noteworthy that all of these diverse substitutions with the exception of [Tyr7] and [Pra7] yielded analogues (15 and 16, respectively) with increased IC50 and significantly shorter duration of action than degarelix. The rationale for the introduction of Asn, Asn(Me) and Asn(Me)2 was to increase the potential for hydrogen bond formation. While Pra in 16 could induce an intra- or intermolecular π-π interaction with aromatic groups in degarelix or the receptor, respectively, the sulfhydryl of cysteine in 17 could be used for further derivatization at position 7. While 16 was the most potent analogue in vitro of this series, (IC50 = 2.11 nM), the antagonist potency of 17 was almost 100 times less (IC50 = 178.2 nM). The poor affinity of 17 was confirmatory of an earlier study (unpublished results) whereby a partial cysteine scan of degarelix had shown loss of activity when introduced at positions 1, 2 and 3. Overall, our observations are complementary to those of Millar et al.35 and Hocart et al.36 who observed a limited preference for hydrophobic residues at position 7. Isopropyl-ornithine (IOrn) and isopropyl-lysine (ILys) were first introduced in GnRH antagonists at position 8 by Ljungqvist et al.7 to yield [Ac-d-2Nal1,d-4Cpa2,d-3Pal3,d-Lys( Nic)6,IOrn/ILys8,d-Ala10]GnRH. They found that the IOrn-containing peptide was less potent than the ILys-containing one, both in an antiovulatory assay and in a histamine release assay. Whereas these two substitutions i.e., IOrn8 in 18 and ILys8 in degarelix (2) have non-significant effect on antagonist potency (IC50s = 1.72 and 1.64 nM, respectively), the former is not as long acting as degarelix. However, analogue 18 is longer acting than Azaline B, Figure 2. It is rather intriguing that such a subtle modification (shortening the chain length by an additional methylene) in 18 and 2 may have an effect on their ability to release histamine (by analogy with Ljungqvist's observation, see above), and to influence those parameters responsible for the duration of action (present investigation). This is further documented in another series whereby the side chains of Lys, Orn, Dab and Dap are each extended by an isopropyl-glycine residue to yield analogues 19–22. The antagonistic potency for all of these analogues 19–22 is equal to that of degarelix, however, the duration of action is extended from short (for 19) to intermediate (for 20) to long*(for 21) to revert back to intermediate (for 22), as if an optimal chain length had been reached in 21 somewhat independently from the actual hydrophobicity of the peptide as measured by the retention time on HPLC at physiological pH (retention time, tR = 30.6, 30.1, 30.3 and 30.6 min for 19–22, respectively). In this series, 21 had a longer duration of action than azaline B and inhibited LH (>80%) release for >96 h while still being shorter acting than degarelix. To further probe the steric environment of the pharmacophore at residue 8, we introduced the cyclohexyl substituent instead of the isopropyl group in 23. Whereas the antagonistic potency of 23 is identical to that of degarelix, the duration of action was significantly altered. Analogue 23 has duration of action similar to that of azaline B but it is about half as long acting as degarelix. Interestingly, 23 may be significantly more hydrophobic than degarelix (measured by the retention time on HPLC, tR for 23 = 31.7 min versus 27.9 min for degarelix). Short duration of action may be due to several factors among which one should consider the rapid release of the peptide from the injection site exhausting all bioactive material or, alternatively insufficient release (due to gel formation) after an initial burst resulting in the retention of a fraction of the material at the site of injection. Although degarelix levels were measured and shown to be above 1.6 ng/mL for the observed duration of gonadotropin inhibition in ovariectomized rhesus monkeys, similar studies were not carried out on shorter acting analogues thus precluding us from any conclusion.37 Additional analogues (24, 25) with the cyclohexyl substitution and otherwise analogous to 21 and 22 are significantly shorter acting. In 26 and 27, we have two analogues differing by one methyl group on the β-nitrogen of Dap8. Whereas both analogues are potent (IC50 ca. 2.0 nM each), the Nβ-methylated 27 is shorter acting than its corresponding non-methylated 26. Another subtle structural difference such as that found in 21 (with (IGly)Dab8) as compared to 26 (with (IβAla)Dab8) with the same composition yield compounds with different and difficult to understand in vivo activities; while analogue 21 is active for >96 hours, analogue 26 is shorter acting by approximately 24 hours. Does this reflect yet to be identified inter- or intra-molecular interactions that significantly alter one or several physicochemical parameters such as enzymatic resistance, solubility, ability to gel or clearance rates, among others? On the other hand, two analogues with the same composition but with a quite different side chain configuration at position 8 such as 20 (Nδ-(IGly)Orn8) and 27 (Nβ-(IβAla)-Nβ-methyl-Dap8) both have an intermediate duration of action. NMR studies in progress will try to address such observations that may be critical for a rational design of optimized structures. In an attempt to further increase the number of inter- or intra-hydrogen bonding opportunities, three additional analogues, 28 (with Nβ-(ICbm)Dap8)), 29 (with Nγ-(ICbm)Dab8) that contain an alkylated carbamoyl function and 30 with an l-hydroorotyl group (Nδ-(l-Hor)Orn8) at position 8 were synthesized; all showed antagonist potency similar to that of degarelix in vitro and short duration of action in vivo. In our previous paper describing the design of degarelix (FE200486) we investigated the effect of Nα-methylation at position 5, which was deleterious (loss of duration of action). Here we scanned positions 6, 7 and 8 with the introduction of an Nα-methyl group in analogues 31–33. Whereas it was well documented that Nα-substitution at position 7 is well tolerated in both agonists and antagonists of GnRH,38,39 Nα-methylation of ILys in azaline B was reported to be detrimental to biological activity in a rat antiovulatory assay.27 As shown here, such a substitution resulted in a shortened duration of action when introduced at position 6 of degarelix and reduced antagonistic potency significantly when substituting ILys (IC50 = 52.24 nM) at position 8.

As we probe closely related structures of degarelix, a promising drug candidate that can be administered sc to achieve long duration of action (>30 days in human),40 we modulated the steric boundaries, ionic properties and hydrogen bond forming capacity of the pharmacophore at positions 3, 7 and 8 of degarelix known to be critical yet not necessarily optimized in analogues reported so far.14,22,27,41 To generate degarelix analogues mono N-alkylated at position 3 we developed an indirect two-step reductive alkylation protocol of the orthogonal amino group of d-Dap/d-Dab residues in otherwise protected resin-bound peptides with various aldehydes thereby generating a wide range of basic amino acids having unique aromatic, steric and electronic characteristics. Concomitantly, we probed the effect of novel substitutions to influence solubility characteristics favorable for slow release formulations comparable to those used for GnRH agonists and which may depend primarily on the hydrophilic character of the analogue. Interestingly, some early generation analogues of degarelix14 including azaline B11 and acyline27 had a significant tendency to gel upon standing in aqueous buffers such as 5% mannitol at concentrations as low as 2.5 mg/mL. Very few of the analogues (10, 16 and 23) reported here show such tendency. The highlight of this work is the observation that very minor substitutions at positions 3, 7 or 8 of degarelix aimed at modifying the steric, hydrogen bonding potential or the ability to form π-π interactions resulted in all cases to loss of duration of action with retention in most cases of antagonistic potency thus paving the way for promising structural studies. Experimental Section Instruments The optical rotation was measured on a Perkin Elmer model 241 polarimeter in a 1 dm microcell at 25 °C at the concentration indicated (w/v %). Thin-layer chromatography (TLC) was performed in a solvent-vapor-saturated chamber on Merck silica gel 60 F254 plates using the following solvent systems: (A) EtOAc:hexane = 1:1, (B) CHCl3:MeOH = 9:1, (C) n-butanol:AcOH:H2O = 4:1:2, (D) CHCl3:MeOH:AcOH = 95:5:3. The plates were visualized by UV, I2, and ninhydrin spray. 1H NMR spectra were recorded on a Varian Mercury 200 MHz spectrometer with CDCl3 as a solvent. Chemical shifts (δ) are expressed in parts per million relative to internal standard tetramethylsilane (TMS). Mass spectra of the amino acids were recorded on a Bruker Esquire 3000 plus instrument using nitrogen/helium gas ESI-MS. Analytical RP-HPLC were run on a system using two Waters 501 HPLC pumps, a Schimadzu SPD-6A UV detector, Rheodyne Model 7125 injector, Omniscribe (Houston Instrument) chart recorder and a Vydac C18 column (0.46 × 25 cm, 5 µm particle size, 300 Å pore size). Preparative RP-HPLC was accomplished on a Vydac C18 preparative cartridge (5 × 30 cm, 15–20 µm particle size, 300 Å pore size) on a Waters Prep LC 4000 preparative chromatograph system with a Waters 486 tunable absorbance UV detector and Omniscribe (Houston Instrument) chart recorder. Matrix assisted laser desorption ionization mass spectroscopy (MALDI-MS) of the peptide analogues were measured on an ABI-Perseptive DE STR instrument. The instrument employs a nitrogen laser (337 nm) at a repetition rate of 20 Hz. The applied accelerating voltage was 20 kV. Spectra were recorded in delayed extraction mode (300 ns delay). All spectra were recorded in the positive reflector mode. Spectra were sums of 100 laser shots. Matrix alpha-cyano-4-hydroxycinnamic acid was prepared as saturated solutions in 0.3% trifluoroacetic acid in 50% acetonitrile. The observed monoisotopic (M + H)+ values of each peptide corresponded with the calculated (M + H)+ values. The capillary zone electrophoresis (CZE) analysis of peptides was performed on a Beckman P/ACE System 2050 controlled by an IBM Personal system/2 model 50Z; field strength of 15 kV at 30 °C using buffer, 100 mM sodium phosphate (85:15, H2O:ACN), pH 2.50, on a Agilent µSil bare fused-silica capillary (75 µm i.d. × 40 cm length). Starting Materials Most amino acid derivatives were obtained from Reanal Finechemical Co. (Budapest, Hungary), including Boc-d-Ala, Boc-Asn(Xan), Boc-Cys(4-MeOBzl), Boc-His(Dnp), Boc-Leu, Boc-Lys(Cbz), Boc-Lys(Fmoc), Boc-Orn(Fmoc), Boc-Pro, Boc-Ser(Bzl) and Boc-Tyr(2Br-Cbz). Boc-Pra was purchased from Neosystem Groupe SNPE (France). Fmoc-Dap(Boc) was obtained from Bachem Inc. (Torrance, CA). Boc-d-4Cpa, Boc-d-2Nal, and Boc-d-3Pal were synthesized at the Southwest Foundation for Biomedical Research (under NIH Contract NO1-HD-6-2928) and made available by the Contraceptive Development Branch, Center for Population Research, NICHD. Boc-l- and Boc-d-4Aph(Fmoc) were synthesized according to the published procedure.13 Boc-l- and Boc-d-Dab(Fmoc) and Boc-l- and Boc-d-Dap(Fmoc) were synthesized according to the published procedure.42 The l-isomer of hydroorotic acid was prepared using a published procedure.43 Boc-Asn(Me) and Boc-Asn(Me)2 were prepared according to the procedure reported earlier.44–46 The benzylchloroformate (Cbz-Cl), 3-bromopropionic acid, cyclohexanone, 2-imidazolecarboxaldehyde, isopropylamine, isopropyl isocyanate, 10% Pd/C, 2-pyridinecarboxaldehyde, 2-quinolinecarboxaldehyde, tert-butyl isocyanate, and sodium cyanoborohydride were purchased from Aldrich Chemical Co. (Milwaukee, WI). Nα-cyclohexyl-Gly(Nα-Cbz),47 IGly(Nα-Cbz),48 Boc-Ilys(Nε-Cbz)6 and Boc-IOrn(Nδ-Cbz)6 were synthesized according to the published procedure. The methylbenzhydrylamine (MBHA) resin49 with substitution 0.33 mequiv/g was obtained according to the published procedure of Rivier et al. using p-toluoyl chloride in lieu of benzoyl chloride in the Friedel and Crafts step.50 All solvents were reagent grade or better. Synthesis of Novel Amino Acids l-Nα-Boc-Lys(Nε-cyclohexyl, Nε-Cbz) l-Nα-Boc-Lys(Nε-Cbz) (15.20 g, 40 mmol), cyclohexanone (7.85 g, 80 mmol), MeOH (100 mL), and molecular sieves (6.0 g, 4 Å) were added in a 500 mL parr hydrogenation vessel. The mixture was purged with N2 for 10 min, and then Pd/C 10% (600 mg) was added. The reductive alkylation under H2 at 40 psi was monitored by HPLC for 26 h. After the filtration of the catalyst, molecular sieves and the evaporation of the solvent in vacuum, the desired intermediate l-Nα-Boc-Lys(Nε-cyclohexyl) was obtained as an oil. The l-Nα-Boc-Lys(Nε-cyclohexyl) was cooled to 0 °C and then Cbz-protected using benzyl chloroformate (Cbz-Cl; 8.6 mL, 60 mmol) in a mixture of THF/H20 (1:1, 200 mL), maintained at pH 9.5 by the use of an autotitrator delivering 0.5 N NaOH. The mixture was then stirred at room temperature overnight. The mixture was concentrated under vacuum to remove THF and then extracted with petroleum ether (3 × 100 mL) to remove excess of Cbz-Cl. The pH of the aqueous layer was brought to 2.5 with saturated sodium hydrogen sulfate and then extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with a saturated sodium chloride solution (2 × 100 mL) and dried over anhydrous sodium sulfate. After evaporation of EtOAc under vacuum l-Nα-Boc-Lys(Nε-cyclohexyl, Nε-Cbz) was obtained as a foam. Yield = 11.47 g (24.80 mmol, 62 %); [α]25D = − 5.5 ° (c = 1, MeOH); ESI-MS (M + H)+ = 463.20; (M + Na+) = 485.20; HPLC assay: column (C18 0.21 × 15 cm), buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA in 60% ACN/40% H20, gradient condition: 40–90% buffer B over 50 min at a flow rate of 0.2 mL/min, UV detection: 0.1 20 AUFS at 214 nM, tR = 44.3 min; purity = 98%; 1H NMR (CDCl3) δ 1.15–1.40 (m, 15H, CH3s + CH2s-cyclohexyl), 1.45–1.56 (m, 4H, γCH2-Lys + δCH2-Lys), 1.58–1.92 (m, 6H, βCH2-Lys + CH2s-C-Cylohexyl), 3.10 (t, 2H, δCH2-Lys), 3.68 (m, 1H, CH-Cyclohexyl), 4.28 (m, 1H, αCH-Lys), 5.12 (s, 2H, CH2-Cbz), 6.19 (br, 1H, NH), 7.32 (br s, 5 H, ArHs-Cbz), 9.30 (br, 1H, COOH). βAla(Nα-isopropyl, Nα-Cbz) Isopropylamine (11.90 g, 200 mmol) in absolute ethanol (100 mL) was added dropwise to the stirred solution of 3-bromopropionic acid (15.3 g, 100 mmol) in absolute ethanol (100 mL). After the addition, the mixture was slowly heated and refluxed for 2 h. The reaction mixture was cooled to room temperature and then NaOH (250 mmol) in H2O (100 mL) was added. The excess of isopropylamine and ethanol was distilled off in vacuum. The residue was dissolved in a mixture of THF/H2O (1:1, 200 mL) and Cbz-protected using the procedure similar to that used for l-Nα-Boc-Lys(Nε-cyclohexyl, Nε-Cbz) to yield the desired βAla(Nα-isopropyl, Nα-Cbz) as a thick oil. Yield = 19.90 g (75 mmol, 75%); ESI-MS (M + H)+ = 266.10, (M + Na+) = 288.10; HPLC assay: column (C18 0.21 × 15 cm), buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA in 60% ACN/40% H20, gradient condition: 40–90% buffer B over 50 min at a flow rate of 0.2 mL/min, UV detection: 0.1 AUFS at 214 nM) tR = 13.6 min; purity = 99%; 1H NMR (CDCl3) δ 1.15 (d, 6H, CH3s), 2.64 (t, 2H, CH2-CO), 3.44 (t, 2H, CH2-N), 4.30 (br, 1H, CH-(CH3)2), 5.15 (s, 2H, CH2-Cbz), 7.32 (br, 5H, ArHs-Cbz), 11.20 (br s, 1H, COOH). Peptide Synthesis All of the peptides were synthesized manually by solid-phase peptide synthesis (SPPS) methodology12 using previously described tert-butyloxycarbonyl (Boc) strategy on methylbenzhydrylamine (MBHA) resin (approximately 1 g of starting resin per peptide). Trifluoroacetic acid (TFA) treatment was used for Boc removal for 20 min. Two to three fold excess of protected amino acid based on the original substitution of the resin was used for coupling for 90–120 min. N-terminal acetylation was performed by using excess acetic anhydride in DCM for 15 min. Compounds 1– 2 were synthesized and purified as previously described.13,14 Synthesis of [IOrn8]degarelix (18) Analogue 18 was derived from the fully protected [Boc-d-4Aph(Nω-Fmoc)-Leu-IOrn(Nδ-Cbz)-Pro-d-Ala]-MBHA resin (1 g; 0.33 mmole/substitution). The individual amino acids were incorporated in a sequential manner utilizing either DIC (N, N’-diisopropylcarbodiimide) or TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-hexamethyluronium tetrafluoroborate) mediated activation of the carboxyl group. The extent to which individual couplings had proceeded was qualitatively determined by the ninhydrin test as described by Kaiser et al.51 The Nα-Boc group was removed after each coupling cycle by treatment of the growing peptide-resin with 60% TFA in DCM in the presence of 1% m-cresol for 20 min. The Nα-Boc protected, resin-bound pentapeptide was treated with 30% piperidine in NMP (20 min) to selectively liberate the ω-amino function of d-4Aph. Carbamoylation of the free Nω-amino function of d-4Aph was then carried out using tert-butyl isocyanate (2 mmol) in dry DMF (5 mL) at rt for 12 h. The completion of reaction was monitored by ninhydrin test. After the removal of Nα-Boc from d-4Aph, the synthesis on the resin was continued with the coupling of Boc-4Aph(Nω-Fmoc). The ω-amino function of 4Aph was freed with 30% piperidine in N-methylpyrrolidone (NMP) and then l-hydroorotic acid (1 mmol) was coupled with DIC (1 mmol) and HOBt (1 mmol) in NMP. The mixture was agitated at rt for 2 h and a ninhydrin test indicated the completed reaction. After the removal of Nα-Boc from 4Aph5 at position 5, chain elongation with the four N-terminal amino acids and acetylation gave the fully protected resin-bound peptide precursor. HF treatment (anhydrous) at 0–5 °C in the presence of anisole (10% v/v) yielded the desired crude analogue after elimination of HF under vacuum, diethyl ether wash, extraction with 0.1% TFA in 40% acetonitrile (ACN)/H2O and lyophilization. The crude peptide (0.45 g) was purified by preparative RP-HPLC.52 The peptide was dissolved in 0.25 M triethylammonium phosphate (200 mL), pH 2.25 (TEAP 2.25) and loaded onto the cartridge described earlier. The peptide was eluted using a flow rate of 100 mL/min with a mixture of A (TEAP 2.25) and B (60% ACN, 40% A) and an appropriate gradient (35% B for 10 min followed by a 90 min linear gradient to 65% B). The collected fractions were screened by analytical RP-HPLC under isocratic conditions, with a mixture of A (0.1% TFA) and B (60% ACN, 40% A) at a flow rate of 1.0 mL/min. Appropriate fractions were then combined (diluted 1:2 with water) and desalted on preparative HPLC with a mixture of A (0.1% TFA) and B (60% ACN, 40% A) using a gradient: 35% B (10 min) followed by a 40 min gradient to 75% B. Yield of 18 after purification was 80 mg (47.88 µmol, 14.52%). Analogues 11–17 and 23 were obtained in comparable yields using this general procedure with the appropriate amino acids introduced at position 7 or 8 during the chain elongation step of the resin-bound peptide. The dinitrophenyl (Dnp) protection of the imidazole of histidine in 14 was removed with thiophenol/NMP at the end of the synthesis and prior to HF cleavage. Synthesis of [Nβ, Nβ-di-(2-pyridyl-methyl)d-Dap3]degarelix (3) The fully protected [Ac-d-2Nal-d-4Cpa-d-Dap(Fmoc)-Ser(Bzl)-4Aph(l-Hor)-d-4Aph(Cbm)-Leu-ILys(Nε-Cbz)-Pro-d-Ala]MBHA-resin was synthesized manually on methylbenzhydrylamine resin (1 g, 0.33 mmol/g substitution). The Fmoc side chain protecting group of d-Dap was then removed with 30% piperidine in NMP (20 min). The exposed d-Dap amino group was reductively alkylated with 2-pyridinecarboxaldehyde (16.5 mmol) in the presence of NaCNBH3 (3.3 mmol) in DMF (25 mL) at rt for 3 h. The cycle was repeated with fresh reagents for an additional 3 h. The progress of the reaction was monitored by chloranil test20 (used for detection of secondary amine on resin) and ninhydrin test.51 The completed resin-bound peptide (1.45 g) was then cleaved, washed, extracted and purified by preparative RP-HPLC using the same conditions described for analogue 18. Yield of 3 after purification was 68 mg (38.80 µmol, 11.77%). Analogue 4 was obtained in comparable yield using this procedure and d-Dab at position 3. Synthesis of [Nβ-(2-pyridyl-methyl)d-Dap3]degarelix (5) This analogue was synthesized the same way as analogue 3 except the deprotected amino group of the side chain of d-Dap was first reacted with the excess of 2-pyridinecarboxaldehyde (20 mmol) in DMF (10 mL) at rt for 4 h. All of the solvents and reagents were drained and the resin was washed sequentially with methanol (2 × 25 mL) and dichloromethane (2 × 25 mL). The resin bound peptide aldamine was agitated for 30 min with solid NaCNBH3 (3.3 mmol) in DMF (15 mL). The reduction step was repeated with fresh reagents for an additional 30 min. The progress of the reaction was monitored by chloranil test.20 The workup and purification of the completed peptide was performed as described above. Yield of 5 after purification was 42 mg (25.30 µmol, 7.66%). Analogues 6–10 were obtained in comparable yields using this procedure, different aldehydes (2-quinolinecarboxaldehyde or 2-imidazolecarboxaldehyde) and d-Dab were substituted at position 3. Synthesis of [Nε-(IGly)Lys8]degarelix (19) First, we synthesized [Boc-Lys(Nε-Fmoc)-Pro-d-Ala]-MBHA-resin on 1 g of methylbenzhydrylamine resin with 0.33 mmol/g substitution. This protected resin-bound peptide was treated with 30% piperidine in NMP (20 min) to free the Nε-amino group of lysine and then IGly(Nα-Cbz) (1 mmol) was coupled by using DIC (1 mmol) and HOBt (1 mmol) in NMP. After the removal of Nα-Boc from Lys, the chain elongation with the seven N-terminal amino acids and acetylation gave the completed peptide-resin (1.60 g). The workup and purification of the completed peptide was performed as described above. Yield of 19 after purification was 120 mg (71.80 µmol, 21.78%). Analogues 20–22, 24–26 and 30 were obtained in comparable yields using this procedure and different amino acid derivatives: Nα-Isopropyl-Gly(Nα-Cbz); Nα-cyclohexyl-Gly(Nα-Cbz); Nα-Isopropyl-βAla(Nα-Cbz); l-Hor) coupled on the orthogonal amino group of Orn/Dap/Dab at position 8, during the chain elongation of the peptide resin. Synthesis of [Nβ-(ICbm)Dap8]degarelix (28) This analogue was synthesized the same way as analogue 19 except the deprotected amino group of the side chain of Dap introduced at position 8 was reacted with isopropyl isocyanate (2 mmol) in dry DMF (5 mL) on the resin. The mixture was agitated at rt for 12 h, and the ninhydrin test indicated a complete reaction. After the removal of Nα-Boc from Dap, chain elongation with the seven N-terminal amino acids and acetylation was carried out on the resin. The completed peptide-resin (1.65 g) was then cleaved and purified by preparative RP-HPLC as described above. Yield of 28 after purification was 110 mg (67.40 µmol, 20.41%). Analogue 29 was obtained in comparable yields using this procedure and Dab was substituted at position 8. Nα-Methylation on the resin A general procedure for the introduction of methyl group at the Nα-amino function of a nascent peptide chain on the resin is described in the literature.53 We have used this methodology for the synthesis of some of the analogues (31–33) presented here. In short, the analogue was built on the MBHA resin up to and including the amino acid that would ultimately contain the Nα-methyl group. Removal of the Nα-Boc group (60% TFA in DCM, 20 min) provided the unprotected Nα-amino function. Alkylation of this primary amino group with 4, 4’-dimethoxybenzhydryl chloride54 in the presence of triethylamine gave the corresponding N-terminal secondary amino function now containing the TFA-labile 4, 4’-dimethoxybenzhyryl group. Methylation of this secondary amine by treatment with 36% aqueous formaldehyde in NMP (3:7) in the presence of excess sodium cyanoborohydride (2 × 40 min), followed by treatment with 60% trifluoroacetic acid in DCM (2 × 20 min) to remove the 4, 4’-dimethoxybenzhydryl group, provided the corresponding Nα-methylated peptide. The coupling of N-protected amino acid to a N-methyl amino acid on the resin is difficult and usually gives low to moderate yield of products, which are often contaminated with unwanted diastereomers. We used bromo-tris-pyrrolidino-phosphonium hexafluorophosphate55 (PyBrop)/DIPEA mediated coupling in NMP to get the desired analogues. Analogue 27 was synthesized by introducing Nα-Fmoc-Dap(Boc) at position 3 during the elongation of the peptide chain followed by Nβ-methylation of Dap as described above on the resin. βAla(Nα-isopropyl, Nα-Cbz) was then coupled to Nβ-methyl of Dap using PyBrop/DIPEA in NMP. After the removal of Fmoc group from Nα of Dap, chain elongation was carried out as described for 28. Peptide Characterization (Table 1) Purity of the peptides was assessed using RP-HPLC and CZE under conditions reported in the legend of Table 1. Composition of the analogues was confirmed by mass spectrometric analysis. Biological Testing. Castrated male rat assays Male Sprague-Dawley rats (180–200 g at the beginning of experiments, n = 5–8) were castrated under ether anesthesia 10 days prior to the start of the experiment. The peptides (500 µg) were dissolved in a bacteriostatic water containing 5% mannitol and 0.6% DMSO. The rats were injected sc with a total dose of 50 µg/rat in 50 µL of aqueous buffer. Blood was sampled from the tail tip (300 µL) at the given times. Plasma LH was determined by RIA using reagents provided by the National Pituitary and Hormone Distribution Program of the NIDDK (Bethesda, MD) with the exception of the second antiserum. NIDDK anti-rat LH S11 serum was used. For each experiment, all plasma samples (vehicle control and tested peptides) were measured in the same RIA. Plasma testosterone levels were determined by radioimmunoassay using kits purchased from Diagnostic Systems Laboratories (Webster, TX). Cell culture Human embryonic kidney cells (HEK293 cells), genetically modified to express a cloned human GnRH receptor (Larry Jameson, Northwestern University, IL) and a luciferase reporter gene under the control of LH alpha subunit promoter,56 were cultured in phenol red free DMEM containing 10% (v/v) FBS, G418 (0.4 mg/mL), Penicillin/Streptomycin solution (100 units penicillin and 100 µg streptomycin per mL medium) and l-glutamine (2 mM). The cells were harvested and plated at 5 × 104 cells per well in a volume of 80 µL per well in white 96 well culture plates. The cells were incubated at 37 °C under 5% CO2 overnight for assay the next day. IC50 determination using the reporter gene assay Each compound was assayed in duplicate at 11 descending concentrations in half log increments. Compounds in 1% DMSO (10 µL), or 1% DMSO alone as a control, were added to the hGnRH receptor expressing HEK293 cells followed by gentle mixing and incubation for an additional 10 min at 37 °C under 5% CO2. Following this, GnRH (10 µL) was added to a final concentration of 1 nM. Plates were then incubated for a minimum of 5 hours at 37 °C under 5% CO2 after which 100 µL of luciferase substrate mix was added to each well. Plates were sealed with Packard Topseal film and luminescence measured on a Molecular Devices Analyst after a 10 minute incubation at rt in the absence of direct light. To derive the IC50, the test compound cps values (minus blank cps) were expressed as a percentage of the control cps values (minus blank cps). The percentage values were plotted against the log of the concentration used, and a curve fitted to the data. An IC50 value was derived by non-linear regression to a 4-parameter logistic equation, [sigmoidal dose-response (variable slope)], using the GraphPad Prism (version 2.01) curve fitting software package. The geometric mean of the IC50 from at least two independent experiments is reported for each compound.

We thank Ron Kaiser and Charleen Miller for technical assistance and William Low and Dr. Wolfgang Fisher for MS analysis. Work at the Salk Institute was supported by NIH grant HD-39899. In vitro biological assays were carried out at FRI Inc. We thank Dr. Judit Erchegyi for critical reading of the manuscript and Debbie Doan for manuscript preparation. JR is the Dr. Frederik Paulsen Chair in Neurosciences Professor. Abbreviations IUPAC rules are used for nomenclature except for the following: - Ac acetyl - ACN acetonitrile - 4Aph 4-aminophenylalanine - Atz 5’-(3’-amino-1H-1’,2’,4’-triazolyl) - Boc tert-butyloxycarbonyl - 2Br-Cbz 2-bromocarbobenzoxy - Bzl benzyl - Cbm carbamoyl - Cbz carbobenzoxy - 4Cpa 4-chlorophenylalanine - CZE capillary zone electrophoresis - Dab α,γ-diaminobutyric acid - Dap α,β-diaminopropionic acid - DCM dichloromethane - DIC N, N’-diisopropylcarbodiimide - DIPEA N, N’-diisopropylethylamine - DMF dimethylformamide - Fmoc 9-fluorenylmethyloxycarbonyl - GnRH gonadotropin-releasing hormone - HF hydrofluoric acid - HOBt 1-hydroxybenzotriazole - Hor l-hydroorotyl - IβAla Nα-isopropyl-βalanine - ICbm isopropylcarbamoyl - IGly Nα-isopropylglycine - ILys Nε-isopropyllysine - IOrn Nδ-isopropylornithine - LH luteinizing hormone - MBHA p-methylbenzhydrylamine - NaCNBH3 sodium cyanoborohydride - 2Nal 3-(2-naphthyl)-alanine - NMP N-methylpyrrolidinone - 3Pal 3-(3-pyridyl)-alanine - Pra propargylglycine - PyBrOP bromo-tris-pyrrolidino-phosphonium hexafluorophosphate - RP-HPLC reversed phase high performance liquid chromatography - RGA Reporter Gene Assay - sc subcutaneous - TBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate - TFA trifluoroacetic acid

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