Discovery of benzimidazo-2-amino-1,3,4-thiadiazole carboxylate small-molecule STAT3 inhibitors for colorectal carcinoma therapy.

To identify potent candidate drugs for colorectal cancer (CRC), a series of N-benzimidazole-1,3,4-thiadiazole-2-amine derivatives featuring diverse ester groups were designed and synthesized, based on our preceding research. Systematic pharmacological evaluation demonstrated their inhibitory effects against the IL-6/JAK/STAT3 signaling pathway and CRC cell lines with constitutively activated STAT3. Among these derivatives, compound L20 demonstrated the most potent anti-proliferative activity against HCT116 (IC50 = 0.45 ± 0.05 μM) and other CRC-relevant cell lines. Mechanistic investigations confirmed that L20 directly binds to STAT3 protein (KD = 6.16 μM), specifically interacting with its SH2 domain. This binding resulted in a dose-dependent suppression of STAT3 phosphorylation at Y705 without affecting total STAT3 protein levels. Furthermore, L20 dose-dependently downregulated both the transcription and expression of cyclin-D1 and c-Myc, two critical downstream effectors of STAT3. Additionally, it induced cell cycle arrest and promoted apoptosis in HCT116 cells in a concentration-dependent manner. Notably, in a murine MC38 subcutaneous xenograft model, L20 administration (20 mg/kg, i.p.) significantly suppressed tumor growth, achieving a tumor growth inhibition rate of 59.8 %. These results highlight the promise of L20 as a novel candidate for CRC therapy and establish a compelling basis for the continued develop of STAT3-targeted interventions against CRC.