In Silico Design, Chemical Synthesis, Biophysical and in Vitro Evaluation for the Identification of 1‐Ethyl‐1H‐Pyrazolo[3,4‐b]Pyridine‐Based BRD9 Binders.

In this work, we report the identification of novel bromodomain‐containing protein 9 (BRD9) binders through a virtual screening based on our developed 3D structure‐based pharmacophore model. The <italic>in silico</italic> workflow here described led to the identification of a promising initial hit (<bold>1</bold>) featuring the 1‐ethyl‐1<italic>H</italic>‐pyrazolo[3,4‐<italic>b</italic>]pyridine motif which represented an unexplored chemotype for the development of a new class of BRD9 ligands. The encouraging biophysical results achieved for compound <bold>1</bold> prompted us to explore further tailored structural modification around the C‐4 and C‐6 positions of the central core. Hence, the design and synthesis of a set of 19 derivatives (<bold>2</bold>–<bold>20</bold>) were performed to extensively investigate the chemical space of BRD9 binding site. Among them, four compounds (<bold>5</bold>, <bold>11</bold>, <bold>12</bold>, and <bold>19</bold>) stood out in biophysical assays as new valuable BRD9 ligands featuring IC50 values in the low‐micromolar range. Noteworthy, a promising antiproliferative activity was detected <italic>in vitro</italic> for compound <bold>5</bold> on HeLa and A375 cancer cell line. The successful combination and application of <italic>in silico</italic> tools, chemical synthesis, and biological assays allowed to identify novel BRD9 binders and to expand the arsenal of promising chemical entities amenable to the recognition of this important epigenetic target. [ABSTRACT FROM AUTHOR]