Regulation of female fertility and identification of future contraceptive targets.

Mammalian reproduction is a complex physiological process involving a tightly regulated hypothalamic-pituitary-gonadal axis and the integration of a diverse array of molecular signals. Oral contraceptives (OCs) were introduced over 40 years ago and have evolved over the years through the discovery of new estrogens and progestins, the development of progestin-only pills and the reduction of the estrogen content in combined OCs. Despite the developments that improved the safety profile of current OCs, adverse metabolic and vascular effects caused by the estrogen component and possible neoplastic effects of OCs remain and, thus, necessitate efforts to develop newer, possibly non-steroidal and non-hormonal, contraceptives. Recent advances in our understanding of ovarian endocrinology, coupled with molecular biology and transgenic technology, have enabled identification of several factors that are functionally critical in the regulation of female fertility. Progress in the area of female reproduction is showing great promise for identifying new contraceptive drug targets. In this article, the authors review the field of female contraception with emphasis on novel targets involved in reproductive function and identified through genomics and proteomics. In addition, the usefulness of these targets for contraception purposes will be discussed.

5-Alkylated thiazolidinones as follicle-stimulating hormone (FSH) receptor agonists.

We prepared analogs of potent thiazolidinone-based follicle-stimulating hormone (FSH) agonists 1, that is, 3 that contained an additional 5-alkyl substituent. This extra substituent was added to reduce synthetic problems that arose during preparation of analogs of 1. These compounds (3) were evaluated in a Chinese hamster ovary (CHO) cell line that expressed recombinant human FSH receptor (FSHR) and a luciferase reporter gene regulated by a cAMP response element (CRE). Selected compounds were also tested on a CHO-cell line that over expressed the FSHR for the ability to induce cAMP production. When the 5-alkyl substituent was a methyl group as in analog 16a, similar FSH activity (i.e., EC(50) = 51 nM, 100% efficacy relative to hFSH) to the analogous 5-hydrogen series compound (e.g., 2) was observed; thus, proving that a small 5-alkyl substituent was well tolerated. New derivatives of 3, in which the potentially hydrolytically labile secondary amide function of 1 (-CONH-) was modified to other moieties (e.g., -CH(2)NH-, -CH(2)S-, and -CH(2)OCONH-), were also prepared and evaluated. These congeners (namely 21, 22, and 24) also displayed good potency in the CRE-luciferase assay.

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Preparation of highly substituted gamma-lactam follicle stimulating hormone receptor agonists.

An unusual combination of Weinreb amidation and Mitsunobu lactam formation was used to prepare highly substituted gamma-lactam analogues of a thiazolidinone follicle stimulating hormone receptor agonist. The analogue synthesis was stereoselective and the final products were chemically stable. Biological properties of the target molecules were nearly identical to those of the lead compound.

Identification and characterization of a selective, nonpeptide follicle-stimulating hormone receptor antagonist.

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-[4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene]-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.

Synthesis of (bis)sulfonic acid, (bis)benzamides as follicle-stimulating hormone (FSH) antagonists.

Screening efforts identified (bis)sulfonic acid, (bis)benzamides (1-3) as compounds that interact with the follicle stimulating-hormone receptor (FSHR) and inhibit FSH-stimulated cAMP accumulation with IC(50) values in the low micromolar range. Structure-activity relationship studies using novel analogues of 1-3 revealed that two phenylsulfonic acid moieties were necessary for activity and that the carbon-carbon double bond of the stilbene sub-series was the optimum spacer connecting these groups. Selected analogues (2, 14, and 50) were also able to block FSHR-dependent estradiol production in rat primary ovarian granulosa cells and progesterone secretion in a clonal mouse adrenal Y1 cell line. IC(50) values for these compounds in these assays were in the low micromolar range. Optimization of the benzoic acid side chains of 1-3 led to gains in selectivity versus activity at the thyroid stimulating hormone (TSH) receptor (TSHR). For instance, while stilbene (bis)sulfonic acid congener 2 was only 10-fold selective for FSHR over TSHR, analogue 50 with an IC(50) value of 0.9 microM in the FSHR-cAMP assay was essentially inactive at 30 microM in the TSHR-cAMP assay.