Rational design and synthesis of a heterocycle scaffold for DNMT3A inhibition

DNA methylation, a crucial epigenetic modification involved in human diseases, is catalyzed by DNA methyltransferases (DNMTs). This study presents a rational design strategy for developing DNMT3A-preferential inhibitors based on a cytosine-containing scaffold. Building on the most potent compound identified from enzymatic activity screening, MOE scaffold replacement was employed to generate a focused virtual library of 5287 analogs. Subsequent drug-likeness and synthetic accessibility filtering narrowed the set to 82 candidates. Among the synthesized derivatives, two lead compounds (6o and 6p) exhibited moderate preferential inhibition of DNMT3A over DNMT1 in enzymatic assays, showing IC₅₀ values around 30 μM against DNMT3A, while only weak or no inhibition of DNMT1 was observed even at high concentrations (320 μM). Molecular dynamics simulations suggested stable binding of the compounds to DNMT3A. In addition, the two compounds demonstrated anti-proliferative effects in HCT116 colorectal cancer cells, with compound 6o showing greater potency. These findings highlight 6o and 6p as promising lead compounds for further development of DNMT3A selective inhibitors.