Design, Synthesis, and Pharmacological Evaluation of Second Generation EZH2 Inhibitors with Long Residence Time

Abstract

Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.

A chemical strategy toward novel brain-penetrant EZH2 inhibitors

“…A similar observation was made by incorporation of a 4-thiomethyl pyridone, recently reported to enhance EZH2 potency of compound 4 (Fig. 1) with increased residence time, 46 to provide compound 6e which exhibited biochemical and cellular potency similar to parent compound 2. Therefore, while the 4-SMe group was able to restore the potency lost due to N-methylation in compound 5, the desirable brain-penetrating properties gained through N-methylation were lost.…”

“…This was surprising since this substitution has been associated with enhanced EZH2 potency through increasing residence time. 46 However, the SAR analysis for high affinity compounds is complicated by the enzymatic assay protein concentration of 5 nM resulting in a floor of the assay of 2.5 nM. For example, incorporation of the N-methyl pyridone into compound 20 displayed a greater than 500-fold loss of potency relative to compound 8 but the exact magnitude of the loss is unclear.…”

Identification and characterization of second-generation EZH2 inhibitors with extended residence times and improved biological activity

“…We recently reported that, quite unexpectedly, the residence time of compound 2, the 4-thiomethyl analog of CPI-1205, ( Fig. 4 , A ), displayed an order of magnitude increase in inhibitor residence time ( 29 ) ( Fig. 4 , B ).…”

“…Further, in this work, we also expand the methodology for k on determination to enable fully quantitative potency assessments. Finally, we expand on the structure-kinetic relationships (SKRs) described previously (29) by showing the universality of the key structural motif to significantly enhance EZH2-inhibitor residence time across all scaffolds tested. The insights from our kinetic studies have enabled us to drive our second-generation series SAM-competitive EZH2 inhibitors to sub-pM biochemical potencies that correlate with enhanced tumor cell killing in vitro and in vivo.…”

“…We recently reported residence time enhancement for EZH2 inhibitors as assessed by a jump dilution method utilizing TR-FRET technology ( 29 ). Herein, we describe the full scope of biochemical approaches we used to kinetically characterize high-affinity, pyridone-containing EZH2 inhibitors (EZH2i).…”