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The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor <b>A366</b>. In an effort to improve the activity and explore the structure–activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the <b>A366</b> series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because <b>A366</b> is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the <b>A366</b> analogs. This resulted in the discovery of diverse selective compounds, among which <b>1s</b> and <b>1t</b> showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.