Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity

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Abstract

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.

Introduction

Steroidal progestins used in combination with estrogens in oral contraceptives represent one of the most widely used clinical therapies. The potential therapeutic value of progesterone receptor modulators for contraception and a wide variety of gynecological indications is well established [1]. Despite the safety profile of these compounds, there exist at least three areas for potential improvement. First, progestins used as oral contraceptives exhibit a range of androgenic effects that may modify the beneficial effects of estrogens on coronary heart disease [2]. Second, use of some oral contraceptive products can increase the incidence of breakthrough bleeding, which is a nuisance to women and leads to reduced compliance [3]. Although the cause of breakthrough bleeding is not well understood, it is possible that progesterone agonists with greater receptor and target organ selectivity would provide more acceptable contraceptive options. Third, steroidal compounds are not well tolerated by many patients, therefore, a nonsteroidal progesterone receptor modulator may be preferred in this patient population.

In addition to the potential for improved selectivity of progesterone agonists, there are several areas for potential improvement of progesterone receptor antagonists (PRMs, for progesterone receptor modulators). The contraceptive potential of PRMs has been demonstrated in monkeys by ovulation suppression and prevention of fertilization [4]. In addition, clinical studies have demonstrated the potential of PRMs for treatment of endometriosis and uterine leiomyoma [1], [5], [6]. However, several studies have established that steroidal PRMs possess antiglucocorticoid activity [7], [8], [9]. It is generally accepted that a significant improvement of PRMs would result if this activity were reduced. A PRM with reduced antiglucocorticoid activity would likely provide a promising clinical option for contraception and for treatment of uterine disease. Despite the tremendous potential of these compounds, little progress has been reported in the discovery of selective progesterone receptor modulators.

Nonsteroidal ligands for the estrogen and androgen receptors have been characterized previously [10]. Nonsteroidal compounds generally have improved receptor selectivity relative to their steroidal counterparts. For example, the steroid cyproterone acetate was the first androgen receptor antagonist developed, but it had significant activity on the progesterone receptor. However, Casodex (bicalutamide), a nonsteroidal androgen antagonist, has been shown to be selective for the androgen receptor and possesses neither agonistic or antagonistic activity on the progesterone, glucocorticoid, mineralocorticoid, or estrogen receptors [11]. However, nonsteroidal ligands for the progesterone receptor with the potential to modulate progestin action in women are rare. We have discovered a novel series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines [12], [13] (Combs et al., manuscript in preparation), that demonstrate unprecedented progesterone receptor selectivity and potency in several in vitro and in vivo test models. Tetrahydropyridazines that act as progesterone receptor agonists or as progesterone receptor modulators will be described.

Section snippets

Reagents

Tissue culture medium (RPMI 1640) for T47D cells (American Type Culture Collection, Manassas, VA; ATCC # 45528) and IM-9 cells (ATCC #CCL-159), fetal calf serum and Hepes-buffered sterile saline (HBSS) were purchased from Life Technologies (Rockville, MD). The radioligands [3H]R5020 (progesterone receptor), [3H]R1881 (androgen receptor), [3H]dexamethasone (glucocorticoid receptor) and [3H]diethylstilbestrol (estrogen receptor), as well as unlabeled R5020, were purchased from NEN Life Science

Results

We evaluated over 600 related compounds in the tetrahydropyridazine chemical series for inhibition of [3H]R5020 binding to the progesterone receptor. Initial synthesis of this series of compounds and preliminary structure–activity relationships for binding to the progesterone receptor have been discussed previously [12]. The structure–activity relationships that contributed to high affinity binding have recently been summarized (Combs et al., manuscript in preparation). The lead compound in

Discussion

In these experiments, we have demonstrated that RWJ 26819 and structurally related nonsteroidal progesterone agonists and progesterone receptor modulators display unusual selectivity for the progesterone receptor. In addition, we have shown that activation of the progesterone receptor in vitro by tetrahydropyridazines is markedly reduced compared to steroidal ligands, despite the similar receptor binding affinities. Third, we have shown that the potency of tetrahydropyridazines in vivo compared

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    Present address: Serono Laboratories, 100 Longwater Circle, Norwell, MA 02061, USA.

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