Scaffold structure. The necessity for the lipophilicity of the scaffold is reflected by the gain in potency observed with the thiourea compared to the less lipophilic urea. The analysis of the amino acid residues which surround compound 6 in its pharmacophore binding pose indicates that two hydrogen bonds with Asn152 and one p-p interaction with Tyr155 are established. Recently published docking studies suggest similar interactions for bicyclic substituted hydroxyphenylmethanones. Interestingly, there is a decrease of activity in both compound classes when the hydroxy group is shifted from the meta- to the para position. This similarity in SAR supports the hypothesis that the hydroxyphenyl moieties of both compound classes bind in the same area of the enzyme. In order to evaluate the protein-ligand interactions, the ligands of the different X-ray structures 1316215-12-9 studied were replaced by compounds 6 and 21 according to their pharmacophoric binding modes and the interactions between the inhibitors 6 and 21 and each of the crystal structures were examined. The maximum number of interactions was observed with the crystal structure 1equ, originally containing the inhibitor equiline. The reason for this is the residue Arg258 which protrudes into the active site in case of 1equ. The importance of this amino acid residue was already postulated by Alho-Richmond et al., who proposed to target it in the inhibitor design process. The biological assays employed for the evaluation of inhibitory potency towards 17b-HSD1 and 2 use well established conditions. In the 17b-HSD1 assay, NADH rather than NADPH is used as cosubstrate. Substrate concentrations are adjusted to the corresponding Km-values which are reported in the literature and confirmed by own experiments. Using NADH instead of the more expensive NADPH was found to give comparable results, as mentioned above. The selectivity against 17b-HSD2 should be achieved to mainly avoid systemic effects: This enzyme is downregulated in EDD tissues but is nevertheless present in several organs. However, it is difficult to estimate how high the SF should be to minimize potential side effects due to the lack of respective in vivo data. For our drug development program, an SF of approximately 20 is considered sufficient to justify further biological evaluation. In this study the retroamide 21 is the most 17b-HSD2 selective compound identified. It is Torin 2 striking that the amide 18 shows a complete loss in selectivity against 17b-HSD2. As no 3D-structure of this enzyme is available, an interpretation of this result at protein level is not possible. The data indicate that the orientation of the amide group is an important feature to gain activity for 17b-HSD1 and selectivity against 17b-HSD2. Affinity of the compounds to the ERs wo
