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We have previously found that esters of 11beta-estradiol carboxylates are transformed from an estrogen into an antiestrogen when the 11beta-side chain is increased in length from four to five non-hydrogen atoms (n > or = 5). To understand the structural requirements for this transformation and obtain metabolically stable analogues that are not susceptible to esterase cleavage, we have synthesized other compounds having an 11beta-side chain composed of other functional groups: ketones, amides, ethers, and thiono esters. With the exception of amides, which bind poorly to the estrogen receptor (ER), all of these compounds exhibit antiestrogenic action when the side chain length is n > or = 5. Ethers (n > or = 5), studied in more detail, inhibit the action of estradiol with either ERalpha or ERbeta. In rat uteri they are estrogen antagonists/weak agonists and decrease the concentration of cholesterol in blood (an hepatic estrogenic action). Thus, these short chain and nonpolar 11beta-analogues of estradiol have tissue specific antiestrogenic/estrogenic actions, characteristics of selective estrogen receptor modulators.

Selective estrogen receptor (ER) modulators (SERMs) are important therapeutic agents for breast cancer prevention and treatment. We have synthesized two analogs, E11-2,1 [methyl-(3,17beta-dihydroxyestra-1,3,5(10)-triene-11beta-yl)acetate] and E11-2,2 [ethyl-(3,17beta-dihydroxyestra-1,3,5(10)-triene-11beta-yl)acetate], the methyl and ethyl esters of an estradiol analog, substituted in the B ring at C-11beta with a carboxymethyl group. The shorter methyl ester, E11-2,1, has high ER affinity and high estrogenic potency in the Ishikawa estrogen cell bioassay, whereas the longer ethyl ester, E11-2,2, has even higher ER affinity, but little or no estrogenic activity. We found that this minor change of one methylene group transforms a potent estrogenic agonist into an antagonist in vitro with either ER alpha or beta. In the rat, E11-2,2 acts as a SERM in the uterus, where it inhibits estradiol-induced proliferation, and as an estrogen agonist in the liver and skeleton, where it decreases plasma cholesterol and increases bone growth. The characteristic feature of antiestrogens, including SERMs, is a long and polar side-chain that prevents agonist-induced conformation of helix 12 of ER. E11-2,2 with its short, nonpolar side-chain, lacks this critical structure, presenting the possibility that it might act through a unique mechanism.

We have synthesized derivatives of estradiol that are structurally modified to serve as "soft" estrogens and act within a geographically limited area of the body; estrogens without systemic action. We have previously shown with 16alpha-substituted analogues of estradiol that carboxylates proximal to the steroid ring neither bind to the estrogen receptor nor activate estrogen-responsive genes. However, when the carboxylic acid is masked as an ester, they bind to the receptor and stimulate estrogenic responses. Enzymatic hydrolysis through nonspecific esterases can inactivate these estrogens and thereby limit their area of action. Here, we describe our continued studies to design "soft" estrogens by synthesizing carboxylic acid esters of estradiol at the 7alpha-, 11beta-, and 15alpha-positions in the steroid nucleus at which bulky substituents are accommodated by the estrogen receptor. These compounds were tested for estrogen receptor binding (estrogen receptors alpha and beta), stimulation of an estrogen sensitive gene in Ishikawa cells in culture, and as substrates for enzymatic hydrolysis. Likely candidates were tested in in vivo assays for systemic and local estrogenic action. The biological studies showed that regardless of the point of attachment, all of the short-chain carboxylic acids, C-1 to C-3, were devoid of hormonal action, while many of the esters were estrogenic. The site on the steroid nucleus had great influence on hormonal activity and esterase hydrolysis. Formate esters at 7alpha and 15alpha were good estrogens, but lengthening the chain to acetate dramatically decreased hormonal activity. However, the 7alpha-formate esters were not enzymatically hydrolyzed. At 11beta, the acetate (methyl ester) was an effective estrogen, but increasing the chain length to propionate dramatically reduced hormonal activity. In general, the length of the alcohol from methyl to butyl had only a small effect on receptor binding, and as the size of the alcohol increased, so did esterase hydrolysis. One exception was the 11beta-acetate esters where increasing the alcohol moiety from methyl to ethyl eliminated estrogenic activity (Ishikawa cells) without affecting estrogen receptor binding. Several of the esters were tested in vivo, and two, the methyl and ethyl esters of estradiol-15alpha-formate, appeared to have the requisite properties (high local and low systemic activity) of superior "soft" estrogens.

We attempted to design analogues of estradiol to act as locally active estrogens without significant systemic action. We synthesized a series of 16alpha-carboxylic acid substituted steroids and their esters and tested their action in several assays of estrogenic action, including estrogen receptor (ER) binding, estrogenic potency in Ishikawa cells (human endometrial carcinoma), rat uterine weight (systemic action), and mouse vaginal reductases (local action). All of the estradiol substituted carboxylic acids (formic, acetic and propionic acids) were devoid of estrogenic action. To the contrary, many of the esters had marked estrogenic potency in the receptor and the Ishikawa assays. The esters of the 16alpha-formic acid series had the highest ER affinity with little difference between the straight-chain alcohol esters (from methyl to n-butyl). However, estrogenic action in the Ishikawa assay decreased precipitously with esters longer than the ethyl ester. This decrease correlated well with the increased rate of esterase hydrolysis of longer esters as determined in incubations with rat hepatic microsomes. The most promising candidates, the methyl, ethyl, and fluoroethyl esters of the formate series, were tested for systemic and local action in the in vivo models. All three, especially the fluoroethyl ester, showed divergence between systemic and local estrogenic action. These metabolically labile estrogens will be extremely useful for the therapeutic treatment of the vaginal dyspareunia of menopause in women for whom systemic estrogens are contraindicated.