Synthesis and Structure‐Activity Relationships of <i>N</i> ‐(4‐Benzamidino)‐Oxazolidinones: Potent and Selective Inhibitors of Kallikrein‐Related Peptidase 6

<jats:title>Abstract</jats:title> <jats:p> Kallikrein‐related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high‐throughput screen within the European Lead Factory program. Structure‐guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors <jats:bold>32</jats:bold> ((5 <jats:italic>R</jats:italic> )‐3‐(4‐carbamimidoylphenyl)‐ <jats:italic>N</jats:italic> ‐(( <jats:italic>S</jats:italic> )‐1‐(naphthalen‐1‐yl)propyl)‐2‐oxooxazolidine‐5‐carboxamide) and <jats:bold>34</jats:bold> ((5 <jats:italic>R</jats:italic> )‐3‐(6‐carbamimidoylpyridin‐3‐yl)‐ <jats:italic>N</jats:italic> ‐((1 <jats:italic>S</jats:italic> )‐1‐(naphthalen‐1‐yl)propyl)‐2‐oxooxazolidine‐5‐carboxamide) have single‐digit nanomolar potency against KLK6, with over 25‐fold and 100‐fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, <jats:bold>32</jats:bold> , effectively reduces KLK6‐dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of <jats:bold>32</jats:bold> make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo. </jats:p>