Chemical modification of STL427944 toward morpholine-based 1,3,5-triazines as anticancer agents targeting FOXM1: green synthesis, biological evaluation and ADME-Tox profiling in a colorectal cancer model

Morpholine-based 1,3,5-triazines are a promising chemotype for anticancer drug discovery, particularly through inhibition of transcription factor FOXM1, a key driver of colorectal cancer (CRC) progression. In this study, we developed an ultrasound-assisted, eco-friendly synthesis of 17 new derivatives and evaluated their activity in CRC models. Compared with conventional conditions, the sonochemical protocol offered significantly shorter reaction times, more sustainable solvent use, and improved compliance with Green Chemistry Principles. Preliminary 24-h cytotoxicity screening identified compounds 14 and 15 as the most active. Compound 14 showed IC₅₀ values of 14.9 μM (SW620), 17.5 μM (SW480), and 36.7 μM (CCD841), yielding favorable selectivity indices (2.1-2.5). It was more potent than 5-fluorouracil in SW620 cells (IC₅₀ = 21.7 μM). Functional assays confirmed near complete inhibition of colony formation at 3 μM and suppression of cell migration at 6.25-12.5 μM. Mechanistic studies revealed downregulation of FOXM1 and its downstream effectors CCNB1 and CDC25, resulting in G₂/M arrest and apoptosis. Additional profiling showed weak PI3Kγ inhibition (IC₅₀ = 3.5 μM), high passive permeability (P_e = 11.7 × 10^-6 cm/s), and lower CYP3A4/2D6 interference compared with the reference compounds. The inhibition of colony formation and suppression of cell migration, the key features of the CSC phenotype, suggest that compound 14 effectively targets the populations responsible for self-renewal and metastasis. Its high activity in the CSC-rich SW620 line, including superior potency to 5-fluorouracil, further underscores its therapeutic potential for treating advanced resistant colorectal cancer. In vivo validation using a zebrafish xenograft model demonstrated that compound 14 reduced tumor growth, with the strongest effect observed in the metastatic SW620 line. Overall, compound 14 significantly outperformed the original hit (STL427944), achieving FOXM1 inhibition at <12.5 μM (vs. 25-50 μM for STL). This marks it as a next-generation FOXM1-targeting lead, combining potent and selective anticancer activity with sustainable synthesis, and positioning it as a strong candidate for further preclinical development.