Novel isocryptolepine analogs with submicromolar antiproliferative activity: En route to next-in-class antiestrogens for the treatment of breast cancer.

Abstract

e15166 Background: Estrogen receptor α (ERα)-positive breast cancer (BC) comprises ~70% of cases. Despite advances in endocrine therapy, a lot of patients develop resistance, necessitating novel therapeutic approaches. Indoloquinoline alkaloids, particularly isocryptolepines, represent a promising scaffold. We synthesized and evaluated novel isocryptolepine analogs with enhanced antitumor activity. Methods: Two series of derivatives (n = 27) were synthesized via photochemical cyclization of O -acetyl oximes. Antiproliferative properties and cytotoxicity were assessed by MTT assay (72 h) in BC cell lines MCF7, T47D (ERα+/PR+), HCC1954 (HER2+), and MDA-MB-231 (triple negative), and also in normal cell lines hFB-hTERT, MCF10A. The antiestrogenic activity of hit compound was evaluated using luciferase reporter assay with MCF7/ERE-Luc cells stimulated with 17β-estradiol (E2). The mechanism of action was investigated by western blotting and molecular docking to ERα. Results: Based on the IC 50 values, ranged from 0.24 to 11.4 μM on MCF7 cells, the most and least active indoloquinoline derivatives were isolated. Compound 6d with a fluorine atom at the R 3 position demonstrated pronounced selectivity toward ERα+ BC cells with IC 50 = 0.12 μM (T47D), SI > 25. In luciferase reporter assay 125 nM 6d inhibited ERα transcriptional activity comparable to 4-hydroxytamoxifen (4-OHTam) at the concentration of 100 nM. Molecular docking revealed direct binding of 6d to ERα, with subsequent confirmation of its antiestrogenic activity through western blot analysis, which revealed significant decrease of ERα protein expression at 0.5 μM. Proapoptotic effects, including cleavage of PARP and downregulation of Bcl-2, were also found. Based on structure optimization, series 2 compound 7l, with methyl at R 5 , exhibited 7.4-enhanced antiproliferative potency in MCF7 (IC 50 = 42 nM) compared to hit 6d. Conclusions: We developed novel isocryptolepine analogs with potent ERα-antagonistic properties. Hit 6d demonstrates excellent cytotoxicity, pronounced selectivity for ERα+ BC cells, and dual mechanism of action. These findings warrant further preclinical evaluation of lead 7l, obtained after structure optimization of 6d, as a promising therapeutic candidate for treatment of ERα+ BC and its endocrine-resistant forms. Compound R 3 R 4 R 5 IC 50 values ± SD, μM MCF7 IC 50 values ± SD, μM MDA-MB-231 SI * (ERα– / ERα+) IC 50 values ± SD, μM MCF10A TI ** (norm / ERα+) 6a H H H 1.3 ± 0.1 0.70 ± 0.06 0.54 0.70 ± 0.07 0.54 6d F H H 0.31 ± 0.03 3.4 ± 0.5 11.0 2.3 ± 0.2 7.4 6h H CH 3 H 0.86 ± 0.09 0.68 ± 0.06 0.79 0.67 ± 0.7 0.78 6k F CH 3 H 0.26 ± 0.03 2.0 ± 0.1 7.7 1.5 ± 0.2 5.8 7c F Ac H 11.4 ± 1.1 > 25 - 14.2 ± 1.5 1.2 7l F H CH 3 0.042 ± 0.003 2.4 ± 0.2 57.1 2.9 ± 0.3 69.0 tamoxifen - - - 6.41 ± 0.7 10.8 ± 1.2 1.7 15.6 ± 1.4 2.4 docetaxel - - - 0.0015 ± 0.0003 0.0059 ± 0.0004 3.9 0.0023 ± 0.0004 1.5 * SI – selectivity index, ** TI – therapeutic index.