Yun-Fei Zhu

Discovery of sodium 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}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

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.

Non-Peptide Gonadotropin-Releasing Hormone Receptor Antagonists

Introduction Gonadotropin-releasing hormone (GnRH) is the principal neuroendocrine regulator of the reproductive system in humans. It is a linear decapeptide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NH2, that was first isolated from porcine and ovine hypothalamii. This peptide and related isoforms have subsequently been identified in a wide range of species, indicating that core features of the sequence have been conserved over ∼600 million years of chordate evolution. The GnRH peptide is made by neurons in the hypothalamus and secreted in pulses approximately hourly into the portal blood supplying the pituitary (Figure 1). There, it stimulates secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), into the general circulation. The gonadotropins in turn act at the gonads to support spermatogenesis and synthesis of testosterone in the male and follicular development and production of estrogen and progesterone in the female. Gonadal steroids in turn feed back to regulate the hypothalamus and pituitary. The actions of GnRH are mediated by the GnRH receptor (GnRH-R). It is a member of the rhodopsin family of seventransmembrane receptors and was first cloned from the mouse and subsequently from a variety of other species, including human. It differs from other class A GPCRs by the absence of a C-terminal tail, which is a predominate site of regulation by various kinases in other family members. Binding of GnRH induces a conformational change in the receptor, which in turn activates the GTPases GRq and GR11 resulting in G-protein activation, which stimulates activity of phospholipase C resulting in phosphatidyl inositol turnover, intracellular calcium release, and gonadotropin secretion. Continuous agonist administration causes an initial stimulatory effect, followed by desensitization of the pituitary and eventually down-regulation of gonadotropin secretion over the course of 1-2 weeks. This pituitary down-regulation leads to a profound suppression of the reproductive endocrine axis and can be exploited therapeutically to produce medical gonadectomy which reduces circulating sex steroids to levels equivalent to surgical castration. On the basis of this somewhat paradoxical mechanism, several GnRH agonist peptides such as leuprolide, goserelin, and triptorelin are now commercially available in long acting injectable depot formulations for gonadal suppression. They have found widespread utility for a range of steroid hormone dependent diseases and assisted reproductive therapy as outlined in Table 1. Early studies in the structure–activity relationships of GnRH peptides identified His as critical for receptor activation. Substitutions with D-amino acids at this position and optimization of multiple additional residues in the peptide led to the identification of potent antagonists. These efforts eventually led to multiple peptides that have been evaluated in clinical studies. In contrast to the initial stimulation caused by agonists, antagonists immediately inhibit pituitary gonadotropin secretion. Thus, in patients with advanced prostate cancer, antagonists avoid the initial “flare” in testosterone produced by agonists and result in a more rapid reduction in testosterone. Analogously in women, peptide antagonists reduce uterine fibroid volume in 2-4 weeks, more rapidly than is observed with agonist therapy. Antagonists also require fewer injec* To whom correspondence should be addressed. Phone: 858-617-7740. Fax: 858-617-7696. E-mail: sstruthers@neurocrine.com. a Abbreviations: AUC, area under the curve; b.i.d., twice daily dosing; Cmax, highest concentration; E2, estradiol; ECL, extracellular loop; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; GnRH-R, gonadotropin-releasing hormone receptor; GPCR, G-proteincoupled receptor; GTPase, guanosine triphosphatase; HPG, hypothalamicpituitary–gonadal; im, intramuscular; ip, intraperitoneal; IVF, in vitro fertilization; LH, luteinizing hormone; NTD, N-terminal domain; sc, subcutaneous; TM, transmembrane.  Copyright 2008 by the American Chemical Society

Initial Structure–Activity Relationship Studies of a Novel Series of Pyrrolo[1,2-a]pyrimid-7-ones as GnRH Receptor Antagonists

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.

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists.

The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core structure was a key feature for good receptor binding activity. SAR study of the 6-(4-aminophenyl) group suggests that hydrophobic substituents were preferred. The best compound from this series had binding affinity (K(i)) of 0.4 nM to the human GnRH receptor.

Synthesis and Structure–activity relationships of 1-arylmethyl-3-(1-methyl-2-amino)ethyl-5-aryl-6-methyluracils as antagonists of the human GnRH Receptor

A new class of small molecule GnRH antagonists, the 1-arylmethyl-3-(1-methyl-2-amino)ethyl-5-aryl-6-methyluracils, was designed and a novel stereoselective synthesis for these compounds was developed. The stereochemical integrities of key intermediates (S)-6 and (R)-6 were confirmed by a combination of X-ray crystallography and chiral HPLC determinations. SAR studies were performed, which allowed the identification of derivatives (R)-9f, (R)-9h and (R)-12 as potent hGnRH antagonists (K(i)=20 nM).

Synthesis and Structure–Activity relationships of 1-arylmethyl-3-(2-aminopropyl)-5-aryl-6-methyluracils as potent GnRH receptor antagonists

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.

Zwitterionic uracil derivatives as potent GnRH receptor antagonists with improved pharmaceutical properties.

A novel series of potent zwitterionic uracil GnRH antagonists were discovered that showed reduced liability for CYP3A4 enzyme inhibition.

Quinoline-carboxylic acids are potent inhibitors that inhibit the binding of insulin-like growth factor (IGF) to IGF-binding proteins.

4-benzylquinolines 5, based on a series of isoquinolines 1, were prepared and tested as inhibitors of the IGF/IGFBP-3 complex based on their ability to displace IGF-I from its binding to IGF-binding protein-3. SAR studies on the 6,7-dihydroxy moiety of the quinoline 5a showed that the catecol moiety could be replaced with other functional groups. Computational modeling of the 5a/mini-IGFBP-5 complex revealed the possible binding site of 5a on IGFBP-5.

Nonpeptide Gonadotropin Releasing Hormone Antagonists

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.

Trapping of a Nonpeptide Ligand by the Extracellular Domains of the Gonadotropin-Releasing Hormone Receptor Results in Insurmountable Antagonism

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.