Diarylpropionitrile (DPN) enantiomers: synthesis and evaluation of estrogen receptor β-selective ligands

Abstract

Two estrogen receptor (ER) subtypes, ERα and ERβ, mediate the actions of estrogens in diverse reproductive and nonreproductive target tissues. ER subtype-selective ligands, which bind to and activate these subtypes differentially, have proved to be useful in elucidating which actions of estrogens proceed through ERα vs ERβ. Some of these ligands show potential as novel therapeutic agents. Diarylpropionitrile (DPN), an ERβ selective ligand that we developed, is a chiral molecule, but it has been studied almost exclusively as the racemic mixture (rac-DPN, 1). Herein we report the development of an efficient enantioselective synthesis of the two isomers, R-DPN (3) and S-DPN (2), and we have compared the in vitro ligand binding affinities, coactivator binding affinities, recruitment potencies, and cellular transcriptional potencies of these isomers. Both enantiomers show a very high affinity and potency preference for ERβ over ERα, typically in the range of 80-300-fold. Although the enantioselectivity is only modest (3-4-fold), the R-enantiomer is the higher affinity and more potent isomer. While ERβ can be effectively and selectively stimulated by rac-DPN or by either R-DPN or S-DPN, R-DPN might be the preferred member of this isomeric series for biological studies of ERβ function.

Figure 1. Coactivator Titration Assay to Determine Relative Coactivator Binding Affinity (RCA) Values for rac-DPN, R-DPN and S-DPN

The fluorescent donor SA-Tb-ERα or SA-Tb-ERβ LBD was titrated against increasing concentration of fluorescein-labeled SRC3 NRID fragment (fluorescent acceptor) in the presence of saturating concentrations of rac-DPN, R-DPN, S-DPN or 17β-E₂ (25 μM). The results in Figures 1A and 1B show ligand-specific binding curves of total tr-FRET values vs. log SRC3 concentrations for ERα and ERβ, respectively. The control FRET (representing the diffusion enhanced FRET; the lowest curves in Figures 1A and 1B) was subtracted from the total FRET values, and the resulting specific FRET binding curves are shown in Figures 1C and 1D. Each assay was performed in duplicate as three independent experiments, and the data from a representative experiment are shown. The concentrations of SRC3 at half-maximal binding (EC₅₀) with both ERs in the presence of different ligands were determined by GraphPad analysis of specific FRET binding curves (Figures 1C and 1D). The RCA of ERα or ERβ bound to rac-DPN, R-DPN or S-DPN for SRC3 was determined as the ratio of EC₅₀ with 17β-E₂/EC₅₀ with different DPNs multiplied by 100. The mean ± SD EC₅₀ (from six measurements) and the respective RCA values are reported in Table 2.

Figure 2. Ligand Titration Assay to Determine Relative Recruitment Potency (RRP) Values for rac-DPN, R-DPN and S-DPN

In these assays, recruitment of a sub-maximal concentration of Fl-SRC3 (100 nM) was evaluated as a function of increasing ligand concentrations. Control-corrected specific FRET values are given. The assay was performed in duplicate at least three times, and the data was analyzed as in Figures 1C and 1D. The concentration of each ligand at 50% SRC3 recruitment (EC₅₀) was calculated, and the RRPs of rac-DPN, R-DPN and S-DPN were determined as the ratio of EC₅₀ with 17β-E₂/EC₅₀ with different DPNs multiplied by 100. The mean ± SD of EC_50s and RRPs from six measurements are shown in Table 3.

Figure 3. Determination of the Relative Cellular Potency (RCP) Values for rac-DPN, R-DPN and S-DPN in HEC1 and U2OS cells

HEC-1 (left panels) or U2OS (right panels) cells were transiently transfected with expression plasmids for ERE-luciferase, human ERα (top panels), or ERβ (bottom panels), and the internal control β-gal as described in the experimental procedures. Experiments with U2OS cells also contained an expression plasmid for human SRC3 in addition to the aforementioned plasmids. The β-gal-normalized reporter gene responses measured at different concentrations of 17β-E₂, rac-DPN, R-DPN, or S-DPN are expressed as percent activity of that observed at the highest concentration of 17β-E₂ with ERα or ERβ. Each assay point represents the mean ± SD of three experiments performed in duplicate. The EC₅₀ response of 17β-E₂ with both ERα and ERβ was set equal to 100%, and the RCP values of the DPNs for each ER were calculated as the ratio of EC₅₀ with 17β-E₂/EC₅₀ with different DPNs multiplied by 100, and are provided in Table 4. A β/α RCP ratio greater than 1 indicates greater cellular potency of ligands towards ERβ than ERα.

Scheme 1

Synthesis of S-DPN (2)^a