tetrasubstitution (Figure 11). The higher polarity of the pyrrole relative to the furans and pyrazoles was postulated to account for the lower affinity. The tetrasubstituted furan derivatives, as represented by 21, proved to be the highest-affinity ligands. While all the furans examined exhibited binding selectivity for ERa, the highest selectivity was achieved with tris-phenolic structures. The optimal C3 alkyl group was shown to be either ethyl or propyl. Furan (21) is a high-affinity ERa-selective ligand (RBA ERa = 140%; RBA ERb = 2.9%). Transcriptional activation studies in HEC-1 cells demonstrated that furan (21) was a potent agonist through ERa (EC50 = 0.33 nmol L_ 1) but had no activity on ERb at concentrations up to 1 mmol L_ 1. Comparison of 21 with its monophenol and bisphenol analogs suggested that it binds in an orientation where the C2 phenol mimics the estrogen A-ring. Molecular modeling studies were consistent with this hypothesis.

Solution-phase, parallel synthesis techniques were used to prepare a series of 2-amino-4,6-diarylpyridines as estrogen receptor ligands (Figure 12).74 Substitution of the two pendant aryl rings and alkylation of the 2-amino group were investigated. Although most of the compounds displayed modest binding (Ki> 100 nmol L_ 1), a significant increase in potency was observed for analogs containing a methyl group meta to the phenol. For example, meta-methyl derivative (22) was the most potent compound prepared in this series (Ki ERa: 20 nmol L_ 1; Ki ERb: 110 nmol L_ 1) (compounds were assayed in a scintillation proximity assay using bacterial lysate with overexpressed human estrogen receptor ligand binding domain). Large hydrophobic groups on the 2-amino nitrogen were necessary for increased binding affinity. Unlike many estrogen receptor ligands, the addition of a second para- or meta-phenol led to a dramatic loss of potency (i.e., R1 = H; R2 = ^-OH). This was rationalized based on molecular modeling studies which suggested that the 4- and 6-phenyl rings span a greater distance than the aryl rings of traditional estrogen receptor ligands such as raloxifene; thus introduction of substitution may cause adverse steric interactions. Compounds of this class display interesting functional activity. In a transient transfection assay using human breast carcinoma cells (T47D), aminopyridine 23 was shown to be a full antagonist at ERb (IC50 = 160 nmol L_ 1), but a weak partial agonist at ERa (EC50 = 30 nmol L_ 1, 16% efficacy relative to E2).

A key feature of natural and synthetic estrogen receptor ligands is the presence of an A-ring phenolic group. Tamoxifen, which appears to lack this feature, is in fact metabolized to 4-hydroxytamoxifen, which then regulates transcription via the estrogen receptor. The interaction of the A-ring hydroxyl with Glu-353 and Arg-394 of the receptor is thought to be a key contact for high-affinity ligands. In an effort to identify nonphenolic A-ring isosteres, Minutolo and

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