Potent antagonists of gonadotropin releasing hormone receptors derived from quinolone-6-carboxamides

SAR studies which focused upon the C-6 position of a recently described series of quinolone gonadotropin releasing hormone antagonists are reported. Synthetic access to diverse quinolone-6-carboxamides was achieved via the palladium-catalyzed amino-carbonylation reactions of iodide 4 with various amines. Amides related to 9y were especially potent, functional antagonists of rat and human GnRH receptors.     

A novel synthesis of 2-arylpyrrolo[1,2-a]pyrimid-7-ones and their structure-activity relationships as potent GnRH receptor antagonists

In the process of developing GnRH receptor antagonists, a novel base-catalyzed cyclization of compounds 5a-b was discovered, which led to the formation of the 2-aryl pyrrolo[1,2-a]pyrimid-7-one core structures 6a-b. These intermediates were further modified at positions 1, 2, 4 and 6 to afford a series of potent GnRH antagonists with low nanomolar K(i) values.     

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.     

2-(3,5-Dimethylphenyl)tryptamine derivatives that bind to the GnRH receptor

A series of 2-(3,5-dimethylphenyl)tryptamine derivatives was prepared and evaluated on a rat gonadotropin releasing hormone receptor assay. Some para-substituents on the 4-phenylbutyl side chain attached to the tryptamine nitrogen led to compounds with potent GnRH receptor binding. The study has helped define structural requirements for GnRH receptor binding for the 2-aryltryptamine GnRH antagonists.     

Inhibition of in vivo and in vitro fertilization in rodents by gonadotropin-releasing hormone antagonists

We have examined the effect of two GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH (Nal-Glu) and Ac(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2),D-Trp(3,6)-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 micro l of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% +/- 5% to 100% +/- 0%, whereas in treated oviducts, fertilization ranged from 25% +/- 6% to 73% +/- 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.     

1,3-Disubstituted-2-carboxy quinolones: highly potent and selective endothelin A receptor antagonists

The design, synthesis, and in vitro biological activity of a series of 2-carboxy quinolone antagonists selective for the endothelin A receptor are presented. Introduction of a second acid group in position 3 of the quinolone ring increases dramatically the selectivity for ET(A).     

Identification and initial structure-activity relationships of a novel non-peptide quinolone GnRH receptor antagonist.

Screening of the Merck sample collection for non-peptide compounds with binding affinity for the rat GnRH receptor led to the identification of the substituted quinolone (1) as a lead compound in the search for a non-peptide GnRH receptor antagonist. Substantial improvements in potency (approximately 300 fold) were achieved by addition of an alkyl amine at the 4-position, a 3,5-dimethylphenyl group at the 3-position and 6-nitro-7-chloro-substitution of the 1 H-quinolone core.     

Investigation of the 4-O-alkylamine substituent of non-peptide quinolone GnRH receptor antagonists.

Synthesis and in vitro activity of the enantiomers of quinolone GnRH antagonist (+/-)-1 are reported. Chiral amino alcohols were prepared from the appropriate cyclic D- or L-amino acids by the Amdt-Eistert homologation followed by reduction of the resulting esters. Incorporation of these pharmacophores was achieved via a novel Mitsunobu alkylation of 4-hydroxyquinolones. The key amine pharmacophore for binding to the rat GnRH receptor was most active in the S-configuration. Ring size was not important for potency with 4, 5, 6, and 7-membered ring amines exhibiting similar potency.     

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.     

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.     

Species selectivity of nonpeptide antagonists of the gonadotropin-releasing hormone receptor is determined by residues in extracellular loops II and III and the amino terminus

Efforts to develop orally available gonadotropin-releasing hormone (GnRH) receptor antagonists have led to the discovery of several classes of potent nonpeptide antagonists. Here we investigated molecular interactions of three classes of nonpeptide antagonists with human, rat, and macaque GnRH receptors. Although all are high affinity ligands of the human receptor (K(i) <5 nm), these compounds show reduced affinity for the macaque receptor and bind only weakly (K(i) >1 microm) to the rat receptor. To identify residues responsible for this selectivity, a series of chimeric receptors and mutant receptors was constructed and evaluated for nonpeptide binding. Surprisingly, 4 key residues located in the amino terminus (Met-24) and extracellular loops II (Ser-203, Gln-208) and III (Leu-300) of the GnRH receptor appear to be primarily responsible for species-selective binding. Comparisons of reciprocal mutations suggest that these may not be direct contacts but rather may be involved in organizing extracellular portions of the receptor. These data are novel because most previous reports of residues involved in binding of nonpeptide ligands to peptide-activated G protein-coupled receptors, including the GnRH receptor as well as mono-amine receptors, have identified binding sites in the transmembrane regions.     

Phosphorylation substrates for protein kinase C in intact pituitary cells: characterization of a receptor-mediated event using novel gonadotropin-releasing hormone analogues

The involvement of protein kinase C in the signal transduction of gonadotropin-releasing hormone (GnRH) action was investigated with a GnRH superagonist, partial agonists, and antagonists in intact rat pituitary cells. Exposure of 32P-labeled cells to GnRH or to the superagonist [D-Nal(2)6]GnRH (200 times GnRH potency in vivo) induced the enhanced phosphorylation of 42-, 34-, 11-, and 10-kDa proteins and the dephosphorylation of a 15-kDa protein as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. This effect was blocked in a dose-dependent manner by potent GnRH antagonists. At its maximally effective concentration of 10(-9) M, [D-Nal(2)6]GnRH induced an up to 2 times more pronounced phosphorylation of endogenous substrates than GnRH at 10(-7) M. This was in accord with its ability to cause an 8-fold increase in the translocation of protein kinase C to the particulate fraction vs. 3.4-fold for GnRH. This effect correlated with potency for a series of GnRH agonists ( [D-Nal(2)6]GnRH greater than GnRH greater than [Gly2]LH-RH) and was prevented by GnRH antagonists, as assessed by a novel phorbol ester receptor binding assay and by a standard kinase assay. Downregulation of protein kinase C by prolonged incubation of the pituitary cells with high concentrations of active phorbol esters abolished protein kinase C activity and also prevented the phosphorylation induced by GnRH, or [D-Nal(2)6]GnRH. The same effect was obtained by preincubating the cells with the protein kinase C inhibitor H-7. In this study we identify for the first time physiological substrates for protein kinase C in intact pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.     

Potent nonpeptide GnRH receptor antagonists derived from substituted indole-5-carboxamides and -acetamides bearing a pyridine side-chain terminus.

A pyridine side-chain terminus has been incorporated into the indole-5-carboxamide and indole-5-acetamide series of GnRH antagonists. Potent activity was observed in binding and functional assays. Certain branched or cyclic tertiary amides were identified as preferred in each series. Alkylation of the side-chain secondary amine had generally unfavorable effects. Variations of the gem-dialkyl substituents in the indole-5-acetamide series were also investigated.     

Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists

The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors.     

The discovery of novel small molecule non-peptide gonadotropin releasing hormone (GnRH) receptor antagonists

A novel series of non-peptide derivatives 1, 14, and 15 that bind with high affinity to the human GnRH receptors is discussed. The discovery was made from screening our in-house libraries that contained the active structure 2 along with a trace amount of a second active structure 1 that was derived from an acid-induced rearrangement. From this structure type 1, a series of guanidine and non-guanidine containing analogues were prepared and tested as GnRH receptor antagonists. Compounds derived from this series bind to both human and rat GnRH receptors and antagonize GnRH-mediated increases in inositol phosphate production in cells containing recombinant human receptors. These compounds or their analogues may be useful as therapeutic agents for the treatment of hormone-dependent pathologies including prostate, breast and ovarian cancers.     

2-phenyl-4-piperazinylbenzimidazoles: orally active inhibitors of the gonadotropin releasing hormone (GnRH) receptor

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional therapy has been limited to peptide agonists and antagonists. Recently, small molecule GnRH antagonists have emerged as potentially new treatments. This article describes the discovery of 2-phenyl-4-piperazinylbenzimidazoles as small molecule GnRH antagonists with nanomolar potency in in vitro binding and functional assays, excellent bioavailability (rat %F>70) and demonstrated oral activity in a rat model having shown significant serum leuteinizing hormone (LH) suppression.     

Dissecting autocrine effects on pulsatile release of gonadotropin-releasing hormone in cultured rat hypothalamic tissue

The control of reproductive function is manifested centrally through the control of hypothalamic release of gonadotropin-releasing hormone (GnRH) in episodic events or pulses. For GnRH release to occur in pulses, GnRH neurons must coordinate release events periodically to elicit a bolus of GnRH. We used a perifusion culture system to examine the release of GnRH from both intact hypothalami and enzymatically dispersed hypothalamic cells after challenge with GnRH analogs to evaluate the role of anatomical neuronal connections on autocrine/paracrine signals by GnRH on GnRH neurons. The potent GnRH agonist des-Gly(10)-D-Ala(6)-GnRH N-ethylamide, potent GnRH antagonists D-Phe(2)-D-Ala(6)-GnRH and D-Phe(2,6)-Pro(3)-GnRH or vehicle were infused, whereas GnRH release from hypothalamic tissue and cells were measured. PULSAR analysis of GnRH release profiles was conducted to evaluate parameters of pulsatile GnRH release. Infusion of the GnRH agonist resulted in a decrease in mean GnRH (P < 0.001), pulse nadir (P < 0.01), and pulse frequency (P < 0.05) but no effect on pulse amplitude. Infusion of GnRH antagonists resulted in an increase in mean GnRH (P < 0.001), pulse nadir (P < 0.05), and pulse frequency (P < 0.05) and in GnRH pulse amplitude only in dispersed cells (P < 0.05). These results are consistent with the hypothesis that GnRH inhibits endogenous GnRH release by an ultrashort-loop feedback mechanism and that treatment of hypothalamic tissue or cells with GnRH agonist inhibits ultrashort-loop feedback, whereas treatment with antagonists disrupts normal feedback to GnRH neurons and elicits an increased GnRH signal.     

Synthesis and biological evaluation of piperazinyl heterocyclic antagonists of the gonadotropin releasing hormone (GnRH) receptor

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional antagonism in vitro. Additional structure–activity relationships within this series are reported along with a pharmacokinetic comparison to the benzimidazole-based lead molecule.