Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS).

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS

Structural similitudes between cytotoxic antiestrogen-binding site (AEBS) ligands and cytotoxic sigma receptor ligands. Evidence for a relationship between cytotoxicity and affinity for AEBS or sigma-2 receptor but not for sigma-1 receptor.

1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene (PBPE) is a cytotoxic derivative of the antitumoral drug tamoxifen. PBPE binds with high-affinity and specificity to the microsomal antiestrogen-binding site (AEBS). PBPE, as well as some other high-affinity AEBS ligands, shares structural features with high-affinity and selective sigma receptor ligands in the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class, such as BD1008, which are cytotoxic against tumoral cells. Based on these structural and pharmacological similitudes, we set out to examine whether AEBS and sigma receptors could be related binding sites. We showed that BD1008 had a high affinity for AEBS. However, prototypical sigma receptor ligands were very low-affinity competitors on AEBS. Surprisingly, AEBS ligands displayed a high affinity for sigma-1 and sigma-2 receptor subtypes, showing that AEBS and sigma receptor-binding sites were not mutually exchangeable. Moreover, phenytoin, which is an allosteric modulator of sigma-1 receptor, was a competitive inhibitor of [3H]tamoxifen on AEBS. These results suggest that the tamoxifen-binding site on AEBS and the sigma ligand-binding site on sigma receptors were not identical but related entities. We also showed here that the high-affinity and specific AEBS ligands also bound sigma receptors with high affinity. Moreover, the compounds that were capable of displacing tamoxifen from AEBS were cytotoxic against tumoral cells but not against the AEBS-deficient cell line Rtx-6. These results confirm that AEBS and sigma receptors might belong to the same family of proteins, and that the tamoxifen-binding site might be involved in the cytotoxicity of AEBS ligands and some classes of sigma compounds

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds