Design, synthesis, and biological evaluation of C-10-esterified dihydroartemisinin-cinnamic acid hybrids as anticancer agents against colorectal cancer.

Dihydroartemisinin (DHA) attracts considerable attention as a promising antineoplastic agent against colorectal cancer. However, its suboptimal antitumor efficacy and its C-10 configurational lability driven by hemiacetal-mediated epimerization collectively impede its clinical development. In this context, a library of 52 DHA-cinnamic acid (DHA-CA) hybrid derivatives with defined C-10 stereochemistry was constructed via stereocomplementary esterification: Mitsunobu conditions selectively afforded 10β-configured esters, whereas HATU-mediated acylation preferentially furnished 10α-isomers. Structure-activity relationship (SAR) exploration revealed that structural modifications, including double-bond saturation, the introduction of electron-withdrawing groups, or altered phenyl substitution patterns, substantially attenuated antiproliferative activity. These findings established that a 10β-configured CA (α,β-unsaturated ester linkage) combined with a bulky, electron-donating meta-substituent (e.g., tBu) constituted the optimal pharmacophore. Guided by this SAR framework, compound 12b-meta was identified as the most potent derivative against colorectal cancer cell lines, exhibiting IC50 values of 0.057 ± 0.014 μM (HT-29) and 0.067 ± 0.016 μM (HCT 116), with a selectivity index (SI) exceeding 447 against NCM460 normal colon mucosal epithelial cells. This profile represents a 52- and 86-fold enhancement in potency relative to DHA. Mechanistic studies showed that 12b-meta suppressed colony formation and DNA synthesis, induced G0/G1 arrest, and was accompanied by reduced Cyclin D1/CDK4 expression. Quantitative proteomic profiling revealed downregulation of proteins related to DNA replication and mitochondrial translation, and immunoblot analysis confirmed RRM2 downregulation. Moreover, 12b-meta showed moderate human plasma stability, with 53.4% remaining after 240 min. These findings established the meta-tBu CA-DHA scaffold as a platform for a potential preclinical optimization in colorectal cancer drug discovery.