Aliau and co-workers have prepared a series of steroidal affinity labels by attaching electrophilic groups (Figure 36).120 Evaluation of the ability of these compounds to inhibit the binding of 3H-estradiol irreversibly indicates that nucleophilic sites in the receptor are relatively remote from the steroid backbone. In addition, cysteine residues were determined to play a role in the covalent attachment sites, as determined by modification of the estrogen receptor with methyl methanthiosulfonate.

Steroid analogs have been used by Labaree and co-workers to evaluate the estrogenic effects of local versus systemic administration of 16a-carboxylic esters/acids (Figure 37).121 Small, aliphatic ester derivatives were shown to bind well to the estrogen receptor and have estrogen-like activity in a human endometrial carcinoma cell line (Ishikawa). In all cases, the corresponding carboxylic acids are substantially less active, as are bulkier esters.116 Increasing the distance of the ester functionality from the steroid ring by homologation of the methylene tether reduces the binding affinity. Receptor affinity was also determined for human ERa and ERb. In general, the affinity of the esters to ERb was significantly diminished relative to ERa. In rodent models for determining estrogenic activity, esters exhibited good efficacy when administered locally but poor activity when administered systemically. The authors postulate that these esters act as 'soft estrogens' that are rapidly inactivated by conversion to the carboxylic acid by hydrolytic esterases found near the site of administration.

Hillisch and co-workers have designed highly selective estrogen receptor isotype-selective and potent ligands using the crystal structure of the ERa LBD and a homology model of the ERb LBD (Figure 38).151 These ligands are approximately 200-fold selective. In rats, the ERa agonist induced uterine growth and was bone-protective whereas the ERb agonist did not have an effect on uterus or bone. Simultaneous administration of both agonists to rats did not lead to an attenuation of the ERa-mediated effects on the uterus.

Conjugated equine estrogens (Premarin) have served as starting points for identifying molecules that have the positive attributes of estrogen replacement therapy (ERT) but with diminished estrogenic activity. Conjugated equine estrogens contain sulfate esters of two structural classes of estrogen that include ring B saturated steroids such as E2 as well as ring B unsaturated estrogens (Figure 39) such as equilin, equilenin, 17b-dihydroequilenin, and 17a-dihydroequilenin (17a-DHEqn), among others. In 1991, Bhavnani and Woolover examined a number of ring B unsaturated steroids in their unconjugated form in order to determine their RBAs for the estrogen receptor and their in vivo effects on uterine hypertrophy in ovary-intact rats.122 All of the individual equine components caused an increase in uterine wet weight, with the exception of 17a-DHEqn, which lacked a significant effect at the highest dose tested. More recently, the sulfate ester conjugate of 17b-DHEqn has been shown to lower serum cholesterol,123 increase hippocampal dendritic spine density in rats,124 and improve arterial vasomotor function in macaques.125 Neuroprotective effects for this compound have also been observed.126 Oral treatment with 17a-DHEqn for 5 weeks prevents bone loss in the proximal metaphysis of excised femurs in a dose-dependent manner. In addition, 17a-DHEqn efficaciously lowers total serum cholesterol with an ED50 of <0.1 mpk. Uterine wet weight is significantly increased by 100% and 142% relative to OVX controls at doses of 1 and 10 mpk, respectively. Thus, the efficacy demonstrated by 17a-DHEqn as an estrogen agonist in preventing bone loss and lowering cholesterol is only observed at doses at which agonist effects are also observed in the uterus, as determined by induction of uterine weight gain.

Benzothiophene (Bt) analogs of the equilenins have been evaluated as SERMS (Figure 40). These compound bind to ERa with Ki ranging from 4 to 24nmolL_ 1.127 Weaker relative affinity to ERb is observed in each case. The

ERa agonist

ERa agonist

ERp agonist

Figure 38 Steroidal subtype selective agonists.

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