MR 20492 and MR 20494:: two indolizinone derivatives that strongly inhibit human aromatase

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (K-i/K-m: 1.0 +/- 0.3 and 0.5 +/- 0.3, respectively). The K-i/K-m for MR 20494 is slightly higher than that obtained for fadrozole (0.1 +/- 0.0) and K-i/K-m for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9 +/- 0.8) and MR 20814 (8.1 +/- .7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher K-i/K-m values obtained with equine enzyme (9.0 +/- 0.6 and 6.1 +/- 1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20 +/- 0.03 mu M, 0.20 +/- 0.02 mu M and 0.50 +/- 0.40 mu M for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9 +/- 0.9 mu M) and MR 20814 (10.5 +/- 0.6 mu M). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site. (C) 1999 Elsevier Science Ltd. All rights reserved.