Dot1L Inhibitor EPZ-5676: Synthesis, pharmacokinetic and tissue distribution studies in Sprague-Dawley rats

Histone methyl-transferase Dot1L can methylate histone 3 on lysine 79 (H3K79). Herein we present the chemical synthesis of a cis/trans mixture of a potent Dot1L inhibitor known as EPZ-5676 (2). Upon preparing compound 2, we tested the compound in mixed and non-mixed lineage leukemia cell lines. The two MLL-rearranged cell lines were MV4;11 and Molm14; whereas the two non-MLL-rearranged (control) cell lines were Molt4 and Kasumi. We observed anticipated in vitro activity for compound 2 in these four leukemia cell lines; results illustrating that Dot1L inhibition can trigger cancer cell death.  In addition, we also tested 2 in a new leukemia cell line known as KO99L; KO99L cells have been shown to over express the membrane amino acid transporter known as L-amino acid type 1 (LAT1). Compound 2 was also observed to decrease cell viability in the KO99L cell; albeit at higher concentrations as compared to the MLL-rearranged cell lines. In addition to in vitro experiments, we also performed in vivo experiments in Sprague-Dawley rats.  Intravenous (i.e. orbital sinus dosing) experiments were performed at two different doses (i.e. 1.0 and 2.0 mg/kg). These rat Pharmacokinetic (PK) results indicate that compound 2 has a slow distribution phase, followed by an extended terminal half-life (i.e. 11.2 ± 3.1 h).  Furthermore, tissue distribution experiments demonstrate that 2 predominately distributes to kidney, blood and liver, and to a limited extend, was detectable in brain.