Design, synthesis and evaluation of pyrimidinobenzylamide and pyrimidinothiophenamide derivatives as inhibitors of DOT1L and related epigenetic targets DNMT3a, PRMT4 and other HMTs.

The histone methyltransferase DOT1L (DOT1 like, disruptor of telomeric silencing) is responsible for methylation of H3K79, leading to oncogene transcription, and it is involved in the development of different types of cancers such as MLL-rearranged leukemia (MLL-r, myeloid-lymphoid leukemia). Inhibitors of DOT1L have therapeutic potential. Thus, we present herein the in silico based design and the multi-step synthesis of different series of non-nucleosidic compounds that mimic the S-adenosyl-l-methionine (SAM) cofactor and inhibit DOT1L. The compounds incorporate an aminopyrimidine moiety coupled to a functionalized aryl based on the structure of published DOT1L inhibitors that have entered clinical trials (EPZ-5676, pinometostat). Their DOT1L activity was determined and structure-activity relationships (SARs) were established, leading to the identification of key moieties for the development of DOT1L-selective compounds. To determine their specificity, the activity of the compounds was evaluated on other methyltransferases that also use SAM as a cofactor, such as DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs), including the PRC2 complex, G9a, PRMT1, PRMT4 and PRMT5. We identified compound 19d (IC₅₀ = 8.0 μM) as a DNMT3a inhibitor, and 1n (EC₅₀ = 19.0 μM), 1p (EC₅₀ = 4.8 μM) and 19g (EC₅₀ = 11.0 μM) as PRMT4 inhibitors based on the in silico approach that was employed. The in vitro ADMET profile of the compounds matched with the generally accepted lead-like criteria and encouraged the further optimization of these non-nucleosidic hit compounds.