In this series the rank order of ERa LiSA activity was F ~ H > Cl > CF3 ~ OCH3 > CH3. For both series, activity in the ERa reporter gene assay matched the apparent binding affinity in the LiSA, although the weaker ligands produced incomplete dose-response curves due to the limitation in maximum concentration tested (10 µM). Examination of the substituent effects on ERa activity showed that, although there was a similar rank order in the para-phenol or meta-phenol series, there was no apparent relationship with phenol pKa (H2O) when considering them independently or in combination (Figure ). Thus, phenol pKa alone was not the driver of ERa activity in the LiSA or reporter gene assays for these thieno [2,3-d]pyrimidine ligands. This result contrasts with the structure activity of simple nonsteroidal estrogens para-hexestrol and meta-hexestrol, where relative binding affinity and agonist activity matched the electron-donating properties of the substituents.