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SAR studies of 2-arylimidazolo[1,2- a ]pyrimid-5-ones 10a – m , which were derived from initial lead 3a , resulted in the discovery of a series of potent nonpeptide human GnRH receptor antagonists. Compounds with good potency (e.g., 10e , K i =7.5 nM) were prepared by introduction of a 2-(2-pyridyl)ethyl at the basic nitrogen and a 3-pentyl ester at the 6-position of the bicyclic core.

SAR studies of lead GnRH receptor antagonists 2a and 2b reported earlier resulted in the discovery of compound 10b which showed much higher potency ( K i =4.6 nM, compared with 2b , K i =230 nM) in which the 7-position of the imidazolo[1,2- a ]pyrimidone core was substituted with a methyl group, and the ester at the 6-position was replaced by the 3-methoxyphenyl group.

The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

Drugs that exhibit insurmountable antagonism are proposed to provide improved clinical efficacy through extended receptor blockade. Long-term suppression of the gonadotropin-releasing hormone receptor (GnRHR) is an important therapeutic approach for a number of sex hormone-dependent diseases. In this study, we describe the mechanism and structural components required for insurmountable activity of a GnRHR antagonist. TAK-013 behaves as an insurmountable antagonist at the human receptor (hGnRHR) but as a surmountable antagonist at the macaque receptor (mGnRHR). Mutation of the eight residues that differ between hGnRHR and mGnRHR identified Ser-203 and Leu-300 in extracellular loops (ECL) 2 and 3 of hGnRHR as essential for the insurmountability of TAK-013. Substitution of the corresponding residues in mGnRHR with Ser and Leu (mGnRHR-P203S/V300L) converts TAK-013 to an insurmountable antagonist. In addition, mutation of Met-24 to Leu in the amino terminus of hGnRHR also ablates the insurmountable antagonism of TAK-013. The mechanism of insurmountability of TAK-013 was determined to be governed by its rate of dissociation from the receptor. Although the association rates of TAK-013 to hGnRHR, mGnRHR, and mGnRHR-P203S/V300L do not differ, the dissociation rate half-life correlates closely with the degree of insurmountability observed (169, 9, and 55 min, respectively). Taken together, these data suggest a model of the GnRHR in which ECL2, ECL3, and the amino terminus engage with TAK-013 upon its binding to the transmembrane region of the receptor. These additional interactions form a "trap door" above TAK-013, restricting its dissociation and thus resulting in its insurmountability.

Nonpeptide antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) have been the subject of considerable interest because of their potential as a new class of oral therapeutics for the treatment of sex hormone-dependent diseases and infertility. While many classes of competitive GnRH-R antagonists have been described, we present here the first characterization of an allosteric nonpeptide GnRH-R antagonist. Previously, 5-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ylmethyl)furan-2-carboxylic acid (2,4,6-trimethoxyphenyl)amide (here called Furan-1) had been demonstrated to be a potent GnRH-R antagonist both in vitro and in vivo. Using mutagenesis, the binding sites for Furan-1 and another potent nonpeptide antagonist (NBI-42902) have been mapped and are shown to be adjacent but nonoverlapping. Furan-1 is shown to affect the binding kinetics of radiolabeled peptide agonists as well as radiolabeled NBI-42902, and the kinetic data fit the allosteric ternary complex model. Furan-1 is considerably negatively cooperative with the nonpeptide antagonist and extremely negatively cooperative with the peptide agonist [125I-His5,d-Tyr6]GnRH so that it is nearly indistinguishable from an orthosteric competitive compound. Taken together, these data were used to develop a model of the nonpeptides bound to the GnRH-R binding site consistent with the current data.

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (Ki = 0.56 nm). Tritiated NBI-42902 binds with high affinity (Kd = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca2+ flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

We have investigated the specific interactions of a series thienopyrimidinediones with the gonadotropin-releasing hormone receptor (GnRH-R). Competitive radioligand binding assays were used to determine the effect of several mutants on nonpeptide binding. Distinct interactions were observed in two separate regions: the N-terminal end of TM7 and the C-terminal end of TM6. The effects of mutants at D302((7.32)) and H306((7.36)) suggest that these residues are part of a hydrogen-bond network important for anchoring the nonpeptides. Structure-activity relationships indicated urea substituents on the 6-(4-aminophenyl) group with a trans conformational preference bind with high affinity and are sensitive to D302((7.32)) mutations. Another interaction area was found between the N-benzyl-N-methylamino substituent and L300((6.68)) and Y290((6.58)). These interaction sites facilitated the derivation of a model in which a representative member of the series was docked into GnRH-R. The model is consistent with known SAR and illuminates inconsistencies with previous hypotheses regarding how this series interacts with the receptor.

CC chemokine receptor 7 (CCR7), which regulates the trafficking of leucocytes to the secondary lymphoid organs, has two endogenous chemokine ligands: CCL19 and CCL21. Although both ligands possess similar affinities for the receptor and similar abilities to promote G protein activation and chemotaxis, they share only 25% sequence identity. Here, we show that substituting N-terminal six amino acids of CCL21 (SDGGAQ) for the corresponding N-terminal domain of CCL19 (GTNDAE) results in a chimeric chemokine that exhibits high affinity binding and G protein activation of CCR7. These data demonstrate that despite dissimilar sequences, the amino terminal hexapeptide of these two chemokines is capable of performing similar roles resulting in receptor activation.

Numerous nonpeptide ligands have been developed for the human gonadotropin-releasing hormone (GnRH) receptor as potential agents for treatment of disorders of the reproductive-endocrine axis. While the equilibrium binding of these ligands has been studied in detail, little is known of the kinetics of their receptor interaction. In this study we evaluated the kinetic structure–activity relationships (SAR) of uracil-series antagonists by measuring their association and dissociation rate constants. These constants were measured directly using a novel radioligand, [ 3 H] NBI 42902, and indirectly for unlabeled ligands. Receptor association and dissociation of [ 3 H] NBI 42902 was monophasic, with an association rate constant of 93 ± 10 μM −1 min −1 and a dissociation rate constant of 0.16 ± 0.02 h −1 ( t 1/2 of 4.3 h). Four unlabeled compounds were tested with varying substituents at the 2-position of the benzyl group at position 1 of the uracil (–F, –SO(CH 3 ), –SO 2 (CH 3 ) and –CF 3 ). The nature of the substituent did not appreciably affect the association rate constant but varied the dissociation rate constant >50-fold ( t 1/2 ranging from 52 min for –SO(CH 3 ) to >43 h for –CF 3 ). This SAR was poorly resolved in standard competition assays due to lack of equilibration. The functional consequences of the varying dissociation rate were investigated by measuring antagonism of GnRH-stimulated [ 3 H] inositol phosphates accumulation. Slowly dissociating ligands displayed insurmountable antagonism (decrease of the GnRH E max ) while antagonism by more rapidly dissociating ligands was surmountable (without effect on the GnRH E max ). Therefore, evaluating the receptor binding kinetics of nonpeptide antagonists revealed SAR, not evident in standard competition assays, that defined at least in part the mode of functional antagonism by the ligands. These findings are of importance for the future definition of nonpeptide ligand SAR and for the identification of potentially useful slowly dissociating antagonists for the GnRH receptor.

Peptide agonists and antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) are widely used to treat a range of reproductive hormone related diseases. Recently, nonpeptide, orally available GnRH-R antagonists have emerged from several chemical classes. To understand how a relatively large peptide-binding pocket can recognize numerous nonpeptide ligands, we undertook a systematic mapping of GnRH-R residues involved in the binding of three nonpeptide antagonists. A region composed of the extracellular portions of transmembrane helices 6 and 7, extracellular loop 3, and the N-terminal domain significantly contributed to nonpeptide antagonist binding. However, each molecule was affected by a different subset of residues in these regions, indicating that each appears to occupy distinct, partially overlapping subregions within the more extensive peptide-binding pocket. Moreover, the resulting receptor interaction maps provide a basis to begin to reconcile structure-activity relationships between various nonpeptide and peptide series and facilitate the design of improved therapeutic agents.

1-(2,6-Difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil (6), a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor, exists as a pair of atropisomers in solution, which was detected by NMR spectroscopy, and separable by HPLC. In addition to a (R)-configured benzylamine, there is a second stereogenic element due to the presence of a chiral axis between the substituted 5-phenyl group and the uracil core. The rate constant of the interconversion (k = 5.07 × 10−5 s−1) of these two atropisomers was determined by proton NMR analysis of a diastereoisomer-enriched sample in aqueous solution at 25°C, and the corresponding Gibbs free energy ΔG# of rotation barrier (97.4 kJ mol−1) was calculated using the Eyring equation. The diastereoisomer half-life at physiological temperature (37°C) in aqueous media was estimated to be about 46 min. © 2005 Wiley-Liss, Inc. Chirality 17:559–564, 2005.

A convenient one-pot synthetic route was developed for the preparation of asymmetric 1,3-dialkyl-1,3,5-triazine-2,4,6-triones from readily available alkyl- or aryl-isocyanates, primary amines and N-chlorocarbonyl isocyanate in excellent yields. Subsequent alkylation with N-protected amino alcohols afforded the desired 1,3,5-triazine-2,4,6-triones in good yields. This methodology was applied to the synthesis of a chemical library acting as antagonists of the hGnRH receptor.

Several efficient synthetic routes for 2-, 4-, and 6-aryl-1,2,4-triazine-3,5-diones were developed. Derivatives were synthesized and studied as gonadotropin-releasing hormone antagonists in an effort to understand structure-activity relationships of the monocyclic compounds.

Uracil derivatives were designed and synthesized to avoid atropisomers observed in the 6-methyluracils as antagonists of the human GnRH receptor. Optimization at the 1- and 5-positions of the uracil resulted in potent compounds such as 24 (Ki=0.45 nM).

One subset of the G-protein coupled receptor (GPCR) superfamily is that which is activated by a peptide carrying an obligatory positively charged residue (GPCR-PA(+)). This subclass is exemplified by receptors for melanocortins, GnRH, galanin, MCH, orexin, and some chemokine receptors variously involved in eating disorders, reproductive disorders, pain, narcolepsy, obesity, and inflammation. Using the methods described in this study, a region of chemical property space enriched in GPCR ligands was identified. This information was used to design and synthesize a "test" library of 2025 single, pure compounds to sample portions of this property space associated with GPCR-PA(+) ligands. The library was evaluated by high-throughput screening against three different receptors, rMCH, hMC4, and hGnRH, and found to be highly enriched in active ligands (4.5-61-fold) compared to a control set of 2024 randomly selected compounds. In addition, the analysis suggested that about 7000 compounds will be necessary to complete the sampling of this GPCR-PA(+) ligand-rich region and to better define its borders.

The synthesis of a series of (R)-1-alkyl-3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils is discussed. SAR around N-1 of the uracil was explored, which led to the discovery that an electron-deficient 2,6-disubstituted benzyl group is required for optimal receptor binding. The best compound from the series had binding affinity of 0.7 nM (Ki) for the human GnRH receptor, which was 8-fold better than the 2,6-difluorobenzyl analog.

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33 (137) and Gln5.42 (227), are consistent with the binding pocket described for biogenic amines, while Lys3.26 (130) and Asn7.32 (305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.

Based on the SAR from bicyclic gonadotropin-releasing hormone (GnRH) antagonists such as 6-aminomethyl-7-aryl-pyrrolo[1,2-a]pyrimid-4-ones (5) and 2-aryl-3-aminomethyl-imidazolo[1,2-a]pyrimid-5-ones (6a,b), a series of novel uracil compounds (8) were derived as GnRH antagonists. The synthesis and SAR studies of 6-methyluracils as human GnRH receptor antagonists are discussed herein. Introduction of a small methyl substituent at the beta-position of the N3 side-chain improved the GnRH binding potency by 5-10-fold. Introduction of a methyl group of (R)-configuration at the alpha-carbon of the N-3 side-chain gave a modest improvement in binding affinity over the unsubstituted ethylene analogues. This modification enabled us to make uracil compounds without the labile 2-pyridylethyl motif on the basic nitrogen while still maintained excellent potency against the hGnRH receptor.

Publisher Summary Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 that stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the pituitary. Because of their peptidic nature, current GnRH based therapeutics requires daily injection or implantation of long acting depots, although nasal formulations of some GnRH peptides are also available. This chapter discusses the development of nonpeptide GnRH antagonists which has resulted in a number of distinct chemical approaches to the problem. This chapter discusses eight distinct GnRH antagonists: thienopyridinones and thienopyrimidinones, 3-arylquinolones, 2-aryltryptamines, macrolides, tetralin, pyrrolopyrimidinone and imidazopyrimidinone, and 5-aryluracils. Several representative molecules from these different series have demonstrated oral activity in suppressing luteinizing hormonein rats or monkeys. Recently, at least two small molecule GnRH antagonists have been evaluated in early human clinical trials. Because the well-described therapeutic mechanism of GnRH peptide based drugs is by suppression of the reproductive endocrine axis, the endocrine effects of the nonpeptide GnRH antagonists in these early clinical trials bodes well for their eventual therapeutic utility in a range of human reproductive related diseases.

The novel synthesis and SAR studies of 6-methyluracils as human GnRH receptor antagonists are discussed. Introduction of a small methyl substituent at the beta-position from N3 of the uracil improved the GnRH binding potency by 5- to 10-fold. The best compound from the series had binding affinity of 5 nM (K(i)) to the human GnRH receptor.

A new class of small molecule GnRH antagonists, the 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-ones, was designed and a novel synthesis for these compounds was developed. The synthesis utilizes a base-catalyzed intramolecular cyclization of fluoromethyl pyrimidone 5 to generate the bicyclic core. Amongst the compounds synthesized, we discovered some highly potent GnRH receptor antagonists (e.g., 12, K(i)=9 nM), which showed enhanced stability towards acidic physiological conditions compared to the des-fluoro analogues.

SAR studies of lead GnRH receptor antagonists 2a and 2b reported earlier resulted in the discovery of compound 10b which showed much higher potency ( K i =4.6 nM, compared with 2b , K i =230 nM) in which the 7-position of the imidazolo[1,2- a ]pyrimidone core was substituted with a methyl group, and the ester at the 6-position was replaced by the 3-methoxyphenyl group.

Initial SAR studies on 1-aminomethyl-2-aryl-3-cyano-pyrrolo[1,2- a ]pyrimid-7-one-6-carboxylates as human GnRH receptor antagonists were discussed. 2-(2-Methylaminoethyl)pyridine was discovered to be a key feature for generating active compounds. The best compound from the series had 25 nM ( K i ) binding affinity to human GnRH receptor.

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[delta 3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-bet a Ala] led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-DTrp-Leu-Arg-Asp-Gly- NH2 and Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-D2Nal-Leu-Arg-Pro-Asp -NH2. These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.

An extended retro-inverso modification was introduced at the central six residues of the somatostatin molecule, the region of internal enzymatic degradations. The synthesis of the analog [Ala4,g-Phe6-r-D-Phe7-r-D-Trp8-r-D-Lys9-r-D-Thr10-m-R,S-Phe11]-somatostatin required a unique strategy accommodating the unusual structure. Side-chain protection based on the t-butyl group in combination with Fmoc and Nps α-amino protection was employed. The key component containing the gem-diaminoalkyl residue was generated by an iodobenzene bistrifluoroacetate-mediated reaction. The separation of diastereomers of the cyclic tetradecapeptide in highly pure form was accomplished by high-performance liguid chromatography on a semipreparative scale. The analogs exhibited very low potency in the growth hormone inhibition test in vitro. This is interpreted as the consequence of the complex structural changes created by the extended retro-inverso modification.

Two-dimensional proton nuclear magnetic resonance spectroscopy at 500 MHz has been carried out on the cyclic decapeptide antagonist of gonadotropin-releasing hormone: cyclo-(delta 3-Pro1-D-pClPhe2-D-Trp3-Ser4-Tyr5-D-Trp6-NMeLeu7-Arg8- Pro9-beta-Ala 10). The antagonist exists in two slowly interconverting conformations. All data are consistent with the conclusion that one form has all-trans peptide bonds and the other has a cis beta-Ala10-delta3-Pro1 bond. With the use of sequential assignment methods, chemical shift assignments were obtained for all backbone and side-chain protons of both conformational isomers except for the serine and tyrosine hydroxyl groups and the C gamma, C delta, and guanidinium group protons of the arginine. Temperature dependence of spectral parameters and magnitudes of observed nuclear Overhauser effects support the interpretation that both conformers of the antagonist consist of two beta-turns (type II', D-Trp6-NMeLeu7; type II, delta 3-Pro1-D-pClPhe2) connected by extended antiparallel beta-like strands.