Synthesis and Evaluation of Analogues of <i>N</i>‑Phthaloyl‑l‑tryptophan (RG108) as Inhibitors of DNA Methyltransferase 1

DNA methyltransferases (DNMT) are promising drug targets in cancer provided that new, more specific, and chemically stable inhibitors are discovered. Among the non-nucleoside DNMT inhibitors, <i>N</i>-phthaloyl-l-tryptophan <b>1</b> (RG108) was first identified as inhibitor of DNMT1. Here, <b>1</b> analogues were synthesized to understand its interaction with DNMT. The indole, carboxylate, and phthalimide moieties were modified. Homologated and conformationally constrained analogues were prepared. The latter were synthesized from prolino­homotryptophan derivatives through a methodology based amino–zinc–ene–enolate cyclization. All compounds were tested for their ability to inhibit DNMT1 in vitro. Among them, constrained compounds <b>16</b>–<b>18</b> and NPys derivatives <b>10</b>–<b>11</b> were found to be at least 10-fold more potent than the reference compound. The cytotoxicity on the tumor DU145 cell line of the most potent inhibitors was correlated to their inhibitory potency. Finally, docking studies were conducted in order to understand their binding mode. This study provides insights for the design of the next-generation of DNMT inhibitors

Identification of Novel Inhibitors of DNA Methylation by Screening of a Chemical Library

In order to discover new inhibitors of the DNA methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC₅₀ of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC₅₀ at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficiently reactivated YFP gene expression in a stable HEK293 cell line by promoter demethylation. Their efficacy was comparable to the DNMT inhibitor of reference 5-azacytidine