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We have previously found that esters of 11β-estradiol carboxylates are transformed from an estrogen into an antiestrogen when the 11β-side chain is increased in length from four to five non-hydrogen atoms (n ≥ 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 11β-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 ≥ 5. Ethers (n ≥ 5), studied in more detail, inhibit the action of estradiol with either ERα or ERβ. 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 11β-analogues of estradiol have tissue specific antiestrogenic/estrogenic actions, characteristics of selective estrogen receptor modulators.

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 16α-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 7α-, 11β-, and 15α-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 α and β), 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 7α and 15α were good estrogens, but lengthening the chain to acetate dramatically decreased hormonal activity. However, the 7α-formate esters were not enzymatically hydrolyzed. At 11β, 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 11β-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-15α-formate, appeared to have the requisite properties (high local and low systemic activity) of superior “soft” estrogens.

Ligands for the glucocorticoid receptor labeled with high-energy isotopes are highly desired for their potential applications in nuclear medical studies of the brain where the dysregulation of this receptor system is thought to be involved in various neurodegenerative disorders. Analogues of the glucocorticoid cortivazol have previously been prepared as target compounds for labeling with high-energy isotopes. However, the phenyl rings of arylpyrazoles of this type are not sufficiently activated for nucleophilic substitution reactions that are generally required for the synthesis of radiohalogenated analogues. Since suitably substituted aromatic nitrogen heterocyclic groups are amenable to nucleophilic substitution, the goal of this study was the synthesis of pyridylpyrazolo and pyrimidylpyrazolo analogues similar to cortivazol that could be labeled with radiohalogens in the pyridine or pyrimidine rings. We describe the synthesis of several [3,2-c]pyrazolo steroids containing pyridyl, halopyridyl, and pyrimidyl substituents at the 2‘ position of the pyrazole ring. These compounds were tested for binding to the glucocorticoid receptor and for biological activity in glucocorticoid responsive HeLa cells grown in tissue culture. Of the pyridyl and pyrimidyl derivatives, 2‘-(3-pyridyl)-11β,17,21-trihydroxy-16α-methyl-20-oxopregn-4-eno[3,2-c]pyrazole showed superior activity in both assays and it was used as the basis for the synthesis of several analogues that were halogenated in the pyridine ring. These halogenated compounds were all tested for their binding to the glucocorticoid receptor and for their biological activity. One, a fluorinated compound 2‘-(2-fluoro-5-pyridyl)-11β,17,21-trihydroxy-16α-methyl-20-oxopregn-4-eno[3,2-c]pyrazole had excellent activity, considerably better than the potent glucocorticoid dexamethasone. Most importantly, fluorination was achieved using a nucleophilic exchange reaction, a method that is adaptable to radiolabeling with the positron-emitting isotope fluorine-18. Thus, considering its superior biological activity and adaptability for facile radiosynthesis, this target compound has the potential for imaging of glucocorticoid receptor containing tissues using positron emission tomography.

We attempted to design analogues of estradiol to act as locally active estrogens without significant systemic action. We synthesized a series of 16α-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 16α-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.

We have synthesized several 7α-fluoro (F) and 7α-iodo (I) analogues of 5α-dihydrotestosterone (5α-DHT) and 19-nor-5α-dihydrotestosterone (5α-NDHT) and tested them for binding to the androgen receptor and for their biological activity in an in vitro assay with cells that have been engineered to respond to androgens. The relative binding affinity to the androgen receptor determined in competition assays showed that in the androstane series the fluoro steroids have the highest affinity and that F-17α-CH3-DHT (4) has a higher affinity than 5α-DHT. All other steroids were somewhat less potent than 5α-DHT with F-DHT (2) = I-17α-CH3-DHT (3) ≥ F-NDHT (6) > F-17α-CH3-NDHT (8) = I-DHT (1) ≥ I-NDHT (5) > I-17α-CH3-NDHT (7). The relative biological activity in cells transfected with the androgen receptor and an androgen responsive reporter gene is 4 ≫ 5α-DHT > 2 > 6 > 3 ≥ 1 ≥ 8 ≥ 5 > 7. The iodinated compound, I-17α-CH3-DHT (3), with the highest binding activity was synthesized labeled with ¹²⁵I and was shown to bind with high affinity, Ka = 1.9 × 10¹⁰ L/mol, and low nonspecific binding to the androgen receptor in rat prostatic cytosol. However, when radiolabeled [¹²⁵I]-17α-CH3-DHT ([¹²⁵I]3) was injected into castrated male rats, it showed very poor androgen receptor-mediated uptake into the rat prostate. This was unexpected in light of its superior receptor binding properties and its protection by the 17α-methyl group from metabolic oxidation at C-17. However, the biological potency of I-17α-CH3-DHT (3) was not as high as would have been expected. When I-DHT (1) and I-17α-CH3-DHT (3) were incubated in aqueous media at 37 °C they rapidly decomposed, but they were stable at 0 °C. The fluorinated analogue 4 treated similarly at 37 °C was completely stable. The products of the decomposition reaction of I-DHT (1) at 37 °C were identified as iodide and principally 17β-hydroxy-5α-androst-7-en-3-one. The temperature dependence of this elimination reaction explains the inconsistency between the high binding to the androgen receptor (measured at 0 °C) and the low biological activity, as well as the poor androgen receptor mediated concentration in vivo. The fluorinated analogue F-17α-CH3-DHT (4) has both high affinity for the androgen receptor and high stability in aqueous media. Of the compounds tested, 4 has the highest affinity for the androgen receptor as well as the highest androgenic activity. Thus it is likely that F-17α-CH3-DHT 4 labeled with ¹⁸F will be an excellent receptor-mediated diagnostic imaging agent.

The key step of the synthetic sequence described is the zwitterionic amino‐Claisen rearrangement of the acetal (VI), yielding the hexahydroisoquinoline (VIII).