Mixed lineage leukemia (MLL) is an incurable form of pediatric cancer. Disruptor of telomeric silencing 1-like (DOT1L), a lysine methyltransferase, is a critical enzyme associated with the initiation and progression of MLL-rearranged acute leukemias. Here we report a synthesis of 14 potential DOT1L inhibitors in a collaborative, early drug discovery investigation. Preparation of these inhibitors involved multiple reaction steps and challenging isolation procedures. However, overall yields for these inhibitors were improved from 3-11% up to 36-42% through optimization of reaction conditions and purification processes. Each target compound and >30 new polyaza heterocycles were characterized with 1H-, 13C-, and 2D-NMR spectroscopy and HRMS spectrometry. Four of the prepared compounds demonstrated confirmed inhibitory activity against DOT1L both in nucleosome and whole cell assays conducted at the University of Michigan Medical School. The most potent DOT1L inhibitor exhibited inhibitory activity with IC50 = 1.0 ± 0.1 μM, which was a 40-fold improvement in potency versus the initial hit. Screening against nine other methyltransferases established target selectivity of the most potent inhibitor and revealed another DOT1L inhibitor with modest inhibitory activity against the protein arginine methyltransferase PRMT3, which is associated with unrelated diseases.Phosphonomethyl ether is a functionality widely present in nucleoside phosphonate-containing antiviral therapeutics including the anti-HIV/ anti-HBV drugs Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafinamide (TAF), and Adefovir. Industrial-scale syntheses of these prodrugs require diethyl p-toluene sulfonyloxymethyl phosphonate (DESMP) to install a phosphonomethyl ether fragment via O-alkylation of a nucleobase segment under basic conditions. This step significantly diminishes overall yield due to complications arising from high water solubility of the corresponding phosphonate diester and partial hydrolysis to the monoester during workup. In collaboration with Professor Anthony J. Arduengo, III and the Medicines for All Institute at VCU, we designed and prepared an innovative phosphonate ester, which may prove advantageous over DESMP in the O-alkylation step. Pinacol sulfonyloxymethyl phosphonate esters were synthesized in three steps using inexpensive reagents and solvents. The optimized conditions facilitate reproducible isolation on moderate scale and limit chemical waste. Products from each step were successfully prepared as solids and were characterized with 1H-, 13C-, and 31P-NMR spectroscopy and HRMS spectrometry. Crystal structures of each product were obtained by X-ray crystallography.