Figure 9 Pyrazole estrogen receptor ligands.

Figure 10 4,5- and 1,4-dialkylpyrazole estrogen receptor ligands.

18 19

Figure 10 4,5- and 1,4-dialkylpyrazole estrogen receptor ligands.

R1 (15, RBA = 7.6%), although the brominated derivative (16) also displayed reasonable binding affinity (RBA = 3.3%. In subsequent work, Stauffer and co-workers69 explored the nature of the optimal pyrazole C4 alkyl group (Figure 9). Modifications at this position (R2 = Me, Et, Pr, Bu) demonstrated that binding affinity increased with intermediate-size substituents (Et, n-Pr and z'-Bu). Interestingly, propylpyrazole triol (17), termed PPT, showed not only a high affinity for ERa (RBA = 49%, purified, full-length, human estrogen receptor) but was also 410-fold selective for ERa (RBA ERb = 0.12%). PPTwas shown to activate transcription via ERa in transfected HEC-1 cells, but had no effect on ERb-mediated transcription, making PPT the first reported ERa-selective agonist.

Both molecular modeling and chemical studies were used in an attempt to elucidate the binding mode of PPT and related analogs. Systematic deletion of the phenol hydroxyls suggested a binding preference for a mode where the C3-phenol acts as the estrogen A-ring mimic. Docking studies suggested that the N1-phenyl occupies space corresponding to the E2 C- and D-ring binding pocket. In an effort to rationalize the significant ERa-selectivity, PPT was modeled using an x-ray crystal structure of diethylstilbestrol bound to ERa.70 The pyrazole core and C4-propyl group were predicted to have contacts to Leu-384 in ERa, which corresponds to Met-384 in ERb. Because of the increased size of the methionine residue in ERb relative to the leucine of ERa, the pyrazole is displaced, causing a significant shift in the position of the N1-phenol, the C5-phenol, and the propyl group. This shift in position is thought to account for the considerably weaker binding to the ERb subtype.

The same group of authors further expanded their work on pyrazole-based SERMs by exploring tetrasubstituted pyrazoles with new substitution patterns.71 The 4,5-dialkyl pyrazoles (Figure 10), as exemplified by 18, displayed relatively weak binding affinity to both human, purified ERa (RBA = 3.6%) and ERb (RBA = 0.6%). Compounds with fused 4,5-cycloalkyl substitution were also prepared and likewise were found to have weak binding affinity. The isomeric 1,4-dialkyl pyrazoles were considerably more interesting and were among the highest-affinity pyrazole ligands reported. For example, pyrazole (19) is a high-affinity, nonselective ligand (RBA ERa = 74%; RBA ERb = 71%). It is interesting to note the lack of selectivity in this series since the previously discussed pyrazoles (e.g., PPT) show a distinct preference for ERa.

Having successfully demonstrated the use of pyrazole-based structures as high-affinity estrogen receptor ligands, the researchers working with Katzenellenbogen subsequently explored the synthesis and biological evaluation of ligands with single heteroatom, five-membered heterocyclic cores, such as furans, pyrazoles, and thiophenes.72 Because of synthetic difficulties, the desired tetrasubstituted thiophenes could not be prepared. However, two trisubstituted thiophenes were investigated but these proved to have very weak estrogen receptor binding (the compounds were initially tested using estrogen receptor from lamb uterine cytosol and the higher affinity ligands were further investigated with human purified receptor). This was not unexpected since trisubstituted pyrazoles were previously shown to have weak affinity.73 The one pyrrole prepared (20) also displayed unexpectedly low affinity, in spite of its

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