Naphthalene sulfone

Figure 23 An SERM designed for uterine fibroids.

in the uterus. The effects on the uterus and ovaries were studied in 6-month-old ovary-intact female rats.17 Oral administration of naphthalene sulfone for 35 days at doses of 0.05, 0.5, 1.0, and 3.0 mgkg _ 1 results in a dose-dependent decrease in uterine weight with a maximal inhibitory dose of 1 mgkg_ 1 (51% reduction in uterine wet weight) and an ED50 of 0.15 mgkg_ 1. Morphometric measurements at the 0.5 mgkg_ 1 dose group compared to the control group show that the total area of the uterine wall was significantly reduced by 53% with nearly equal contributions in reduction of endometrial (54%) and myometrial (51%) compartments. These data confirm substantial antagonist activity in both compartments of the uterus in the presence of circulating levels of E2 over several estrous cycles in the rat. The effects on the ovaries in this study were determined by measuring serum E2 levels and histologic evaluation of ovarian cross-sections. Treatment with the naphthalene sulfone results in serum E2 levels that are similar to vehicle-treated animals at doses that exceed the inhibitory effects on the uterus by greater than 60-fold. Statistically significant increases in E2 are observed at the high dose but are within the normal proestrus range for Sprague-Dawley rats. Histological evaluation of the ovaries of the rats treated with the naphthalene sulfone SERM indicates minimal ovarian stimulation relative to untreated controls, i.e., ovarian weights are decreased at doses >0.5 mgkg_ 1, there are no ovarian cysts, and granulosa cell hyperplasia is observed in only a few animals and generally at a mild level. These data collectively indicate that this naphthalene sulfone SERM is a potent uterine antagonist with minimal ovarian stimulation in rats. ERa-selective ligands

SERMs selective for ERa have recently been reported, including the flavanone,98 dihydrobenzoxathiin,99 chromane100 scaffolds (Figure 24). Members of the chromane family have been shown to be highly receptor subtype-selective, while demonstrating potent in vivo antagonism of E2 in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42-day treatment paradigm, and lowered serum cholesterol levels in OVX adult rat models. The dihydrobenzoxathiins class has typically exhibited low to subnanomolar binding to ERa, with 50- to 100-fold selectivity, and as a result of further study, a derivative has been targeted for development as a potential agent for the treatment of osteoporosis. ERp-selective ligands

The discovery in 1996 of another mediator of estrogen activity has further complicated our understanding of estrogen physiology. This novel receptor, now known as ERb to distinguish it from the classical ERa, is highly homologous in the DNA-binding domain (95%), but shares only 55% sequence identity in the C-terminal region responsible for ligand binding, nuclear localization, and ligand-dependent transactivation properties. Indeed, knockout models of each subtype (aERKO and bERKO) have demonstrated some of the unique biological roles played by each receptor.101 Differences in the C-terminal region make possible the discovery and design of ligands with divergent binding affinities and potencies of both agonist and antagonist activity.

Although the overall sequence identity of the C-terminal region is modest between ERa and ERb, the overall structural differences in the ligand-binding pocket are subtle. Three-dimensional structures of human ERb bound to genistein and rat ERb bound to raloxifene have been described.102 In the structure of genistein (45) bound to ERb, the phenolic hydroxyl of genistein interacts with the Glu-Arg-water triad through a hydrogen bond, while the flavone hydroxy at C7 interacts with the distal His at the end of the cavity (Figure 25). The remaining hydroxy at C5, which is presumably hydrogen-bonded to the adjacent carbonyl group, does not interact with the protein. The overall pocket size is slightly smaller in ERb (390 A3 for ERb-genistein versus 490 A3 for ERa-E 2). This reduction in size is mainly due to the substitution of Leu-384 in ERa with Met-336 in ERb. Of the 22 hydrophobic residues that line the pocket, there is only one other amino acid substitution: Met-421 in ERa is Ile-373 in ERb.

Krege and co-workers have described the ERb-selective activity of several naturally occurring phytoestrogens (Figure 26).101 Daidzein (46) and genistein (45) are closely related, differing only by the addition of a hydroxyl group

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