Development of 7-hydroxy-4-methylcoumarin–cyanoacrylate hybrids with antiproliferative activity against breast cancer cells: from molecular design to in vivo validation

Abstract

In the present study, a series of novel 6,8-dibromo-7-hydroxy-4-methylcoumarin–cyanoacrylate hybrids and their corresponding pyrazole derivatives were designed, synthesized, and evaluated for antiproliferative activity against breast cancer cells. Among the tested compounds, cyanoacrylate derivative 8 emerged as the most active analogue against MCF-7 cells (IC50 = 5.9 ± 0.4 µM) and retained antiproliferative activity against MDA-MB-231 cells (IC50 = 11.7 ± 0.4 µM). Compound 8 exhibited weaker cytotoxicity toward the non-cancerous MCF-10A and WI-38 cell lines (IC50 = 46 ± 3 and 37.4 ± 1.3 µM, respectively), although the magnitude of selectivity was cell-context dependent. Mechanistic investigations in MCF-7 cells revealed pronounced G2/M cell-cycle arrest (7.57% to 32.24%), inhibition of tubulin polymerization (IC50 = 4.7 ± 0.2 µM), and EGFR kinase activity (IC50 = 50 ± 3 nM). Flow-cytometric analysis showed an increase in total apoptotic cells from 2.71% to 33.19%, while LDH release increased 2.88-fold, indicating concomitant loss of membrane integrity. qRT-PCR demonstrated upregulation of BAX (7.9-fold), caspase-9 (8.8-fold), and p53 (4.7-fold) and downregulation of BCL-2 (0.27-fold), with corresponding protein-level changes confirmed by ELISA, providing complementary evidence for activation of the intrinsic apoptotic pathway. Molecular docking provided structural hypotheses for potential interactions of compound 8 within the tubulin colchicine-binding region and EGFR ATP-binding site. In vivo, compound 8 significantly suppressed tumor burden in the Ehrlich ascites carcinoma (EAC) mouse model, reducing viable tumor-cell counts by up to 72.4% and ascitic tumor volume by 85.3%. Treatment also ameliorated tumor-associated hepatic and renal dysfunction, oxidative stress, and inflammatory alterations, with histopathological and immunohistochemical findings supporting tissue recovery. Collectively, these findings identify compound 8 as a promising antiproliferative and antitumor lead and support further optimization and evaluation of this scaffold in more clinically relevant breast cancer models.