To confirm the differences in ERa activity, freshly prepared samples of 1 and 2 were tested in a two-hybrid ligand sensing assay (LiSA) that utilizes the recruitment of a specific peptide to the surface of the receptor to measure the ability of steroidal and nonsteroidal estrogens to interact with the ligand-binding domain of ERa. The assay confirmed our previous observation that meta-phenol (2) was >15-fold weaker than para-phenol (1) as an ERa ligand in the LiSA (Table ), a result that was in part due to differences in binding affinity as shown in a radioligand competition binding assay using [ 3 H]-E2 (Figure ). This difference in ERa binding activity in the LiSA translated to ~100-fold weaker agonist activity measured using a standard reporter gene assay in HepG2 cells, with meta-phenol (2) being a much poorer estrogen than paraphenol (1) (Table ). A cocrystal structure of meta-phenol (2) with the ERa ligand binding domain (LBD) revealed that, despite its weaker estrogenic activity, the phenolic oxygen did indeed form hydrogen bonds with E353, R394, and a bound water molecule while the hydrophobic thieno [2,3-d]pyrimidine core occupied the same region of the ligand binding pocket as 1 (Figure ). Thus, although meta-phenol (2) was able to form an intricate network of hydrogen bonds with E353 and R394 that mimicked the binding of the natural steroid hormone E2 within the ERa ligand binding pocket, it was surprisingly less effective as a nonsteroidal estrogen than para-phenol (1) in both the LiSA and reporter gene assays. To shed light on the origin of the differences in ERa binding and activity of the para-phenol (1) and metaphenol (2), several new thieno [2,3-d]pyrimidine analogs were synthesized. An SNAr reaction of 2,4-dichloro-5-phenylthieno [2,3-d]pyrimidine with various benzylamines was used to produce the phenol analogs (Scheme 1). Scheme 1. Synthesis of thieno [2,3-d]pyrimidines (3-13). (i) Amine, CHCl3, 70 ºC, Et3N. (ii) NaBH4, CoCl2•6H2O, THF/H2O (2:1). (iii) NH2OH.HCl, H2O/EtOH (4:1). (iv) AcOH, Zn dust, D.