Three new monosquaramides (3a–c) were synthesized, characterized by IR, NMR and X-ray, and evaluated for inhibitory activity against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. The target compounds inhibited DNase I with IC50 values below 100 μM, being at the same time more potent DNase I inhibitors than crystal violet, used as a positive control. 3-Ethoxy-4-((1-(pyridin-3-yl)propan-2-yl)amino)cyclobut-3-ene-1,2-dione (3c) stood out as the most potent compound, exhibiting a slightly better IC50 value (48.04 ± 7.98 μM) compared to the other two compounds. In order to analyze potential binding sites for the studied compounds with DNase I, a molecular docking study was performed. Compounds 3a–c are among the most potent small organic DNase I inhibitors tested to date.
