# Synthesis, Antitumor Activity Evaluation and Mechanistic Study of Novel Bis‐Heterocyclic Chalcones Against Liver Cancer

## TL;DR

Researchers synthesized new chalcone compounds that show strong anticancer effects on liver cancer cells by inducing cell death.

## Contribution

The synthesis and evaluation of bis-heterocyclic chalcones with potent antitumor activity against liver cancer cells.

## Key findings

- Compound 3f showed the strongest cytotoxicity against Huh-7 and Huh-1 liver cancer cells.
- Compounds 3d and 3f induce apoptosis in liver cancer cells, as confirmed by Western blot and flow cytometry.
- Bis-heterocyclic chalcones are promising structural units for further anticancer drug development.

## Abstract

Chalcones and heterocyclic compounds exhibit remarkably high activity in medicinal chemistry. In recent years, bis‐chalcones have been reported to possess excellent anticancer activity. We synthesized a series of bis‐heterocyclic chalcones via asymmetric chain synthesis, with the aim of making new discoveries in anticancer activity.

Bis‐heterocyclic chalcones were synthesized via Claisen‐Schmidt condensation and alkylation reactions. The inhibitory activities of the synthesized compounds against Huh‐1, Huh‐7, and HepG2 cell lines were evaluated using the CCK‐8 assay. Furthermore, the mechanism of action of these compounds was explored through live/dead cell staining, flow cytometric analysis, and Western blotting experiments.

Twelve bis‐heterocyclic chalcone compounds were synthesized. All synthesized compounds were fully characterized by spectroscopic methods and evaluated for their cytotoxic activities against Huh‐1, Huh‐7, and HepG2 cell lines using the CCK‐8 assay at concentrations ranging from 0 to 100 μM. Among them, derivative 3f exhibited the most potent cytotoxicity against Huh‐7 (IC50 = 8.40 μM) and Huh‐1 (IC50 = 6.75 μM), whereas HepG2 cells were most sensitive to compound 3d (IC50 = 27.99 μM). The mechanisms underlying the antitumor effects of 3d and 3f were further investigated through live/dead cell staining, flow cytometry, and western blot analysis. The results demonstrate that both compounds effectively induce apoptosis in liver cancer cells.

Bis‐heterocyclic chalcone compounds exhibit favorable inhibitory activity against hepatocellular carcinoma cells by inducing cell apoptosis, and thus can serve as a class of pharmaceutically active structural units for further in‐depth research and screening.

Western blotting was performed to analyze the expression of Caspase 3 and Cleaved‐Caspase 3 in Huh1 cells following different treatments, which supports the finding that bis‐heterocyclic chalcones 3d and 3f can inhibit the activity of hepatocellular carcinoma cells by inducing apoptosis.