Na

Figure 27 Benzothiophene compounds.

coupling reaction of benzothiophene boronic acids with aryl bromides and then screened for binding affinity and selectivity at ERb and ERa. The compounds were also evaluated for their ability to activate an estrogen response element in HeLa cells stably transfected with human ERa or ERb receptors using a luciferase reporter gene assay. The binding selectivities for ERb reached 20-fold (R = OH) with binding affinities for ERb of 100 nmol L _ 1 (R = H or F) to 4 mmol L _ 1 (R = Et). The reported agonist activities are good (EC50 = 17nmolL _ 1, R = H; EC50 = 32nmolL _ 1, R = F) but the selectivities are more modest (nonselective to fourfold selective for R = F). The compounds were also assayed for agonist activity in a human neuronal cell line (neuroblastoma SH-SY5Y transfected with human ERb and the luciferase reporter gene bearing an estrogen response element). All compounds retained agonism with an EC50 comparable to that observed in HeLa cells.

Katzenellenbogen's group discovered ERb-selective diarylpropionitrile (54) by screening a select group of compounds for transcriptional activity by ERa and ERb in human endometrial cancer cells ( ). The RBA of diarylpropionitrile (54) is 70-fold selective for ERb and is a full agonist with 170-fold higher relative potency in transcription assays.

A series of analogs were prepared in which the ligand core and aromatic rings of diarylpropionitrile (54) were modified (Figure 28). The aromatic ring analogs lacking either phenolic hydroxy group lost substantial affinity (30- to 60-fold for ERa and 250- to 370-fold for ERb). Repositioning of the phenolic hydroxy groups demonstrated a preference forpra-substitution. Affinity for ERa is lowered twofold if R2 is meta-OH and 10-fold if R1 is meta-OH. For ERb a meta-OH group resulted in a six- to eightfold drop in affinity. When both R1 and R2 are^-OH, the addition of a methyl group ortho to R2 is tolerated; but interestingly, the addition of a methyl group ortho to R1 resulted in a threefold boost in binding affinity for both ERa and ERb. Extension of the nitrile by a single methylene resulted in moderate loss of affinity and a significant decrease in selectivity. Addition of a methyl group a to the nitrile is more tolerated, while addition of an a-ethyl group resulted in a 7- to 10-fold increase in ERb affinity but concurrently diminished ERb selectivity. The preference of the receptor pocket for the antiperiplanar conformation with respect to the aryl rings is demonstrated by a pair of dinitrile diastereomers. The overall affinity of the meso diastereomer, which prefers an antiperiplanar conformation, is 10-fold more than the DL-diastereomer, which prefers the synclinal conformation. ERb selectivity is maintained by these dinitriles. The unsaturated analog of diarylpropionitrile (54) is identical to the parent while the unsaturated dinitrile has 32-fold more affinity for ERa but only eightfold selectivity for ERb. The steric analogs in which the nitrile group is replaced by acetylene or propyne have a 10- to 12-fold increase in binding affinity for ERa and a two- to fourfold increase for ERb relative to the parent nitrile. Thus, the acetylene analogs have better affinity but lower selectivity. The perfluoroalkyl analogs have slightly better affinity but lower selectivity. The polar analogs lose overall binding affinity and ERb selectivity.

The analogs were examined for transcriptional activities in human endometrial cancer cells. All of the nitrile analogs examined were agonists at ERa and ERb, although some low-affinity analogs did not reach full efficacy. In general, nitrile analogs with the highest affinities also had the highest potencies. Compounds 54, 60, 62, 64, and 65 were all more potent and selective than genistein. The steric, perfluoroalkyl, and polar analogs all had only modest transcriptional potency selectivity.

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