Design, synthesis, and biological evaluation of Combretastatin A4 dimers for the treatment of hepatocellular carcinoma.

Combretastatin A4 (CA4) is a potent microtubule inhibitor, but its phenolic hydroxyl group is prone to oxidation into toxic quinone products, leading to poor metabolic stability and dose-limiting toxicity that restricts its clinical application. To overcome this limitation, we propose a novel dimer prodrug strategy that covalently links two CA4 molecules via an enzyme-cleavable linker, achieving temporary masking of the phenolic hydroxyl group. The resulting CA4-dimer1 and CA4-dimer2 exhibited potent in vitro antiproliferative activity. Of the two dimers evaluated, CA4-dimer1 demonstrated superior in vitro potency. Crucially, CA4-dimer1 achieved significant tumor suppression rates in patient-derived xenograft (PDX) liver cancer models without notable toxicity. CA4-dimer2 releases active CA4 through gradual hydrolysis but showed slightly lower in vivo release efficiency compared to CA4-dimer1. Flow cytometry and Western blot analysis revealed that this prodrug exerts antitumor activity through the same mechanism as the parent compound CA4. Collectively, the reported CA4-dimer prodrug strategy offers a highly promising approach for treating liver cancer and enhancing the therapeutic index of phenolic drugs, while also providing a potential strategy for structural optimization of drugs containing phenolic hydroxyl groups.