Background: DNase I inhibitors may be useful as biochemical tools and as potential leads for investigating pathological processes associated with excessive DNA degradation. Squaramides represent attractive scaffolds for drug discovery owing to their rigid, highly polarized structure and versatile hydrogen-bonding properties. Methods: Four novel pyridine-containing squaramides (5a–5d), comprising two symmetric and two asymmetric derivatives, were synthesized and structurally characterized by spectroscopic methods and, for 5a and 5b, single-crystal X-ray diffraction. Their DNase I inhibitory activity and cytotoxicity were evaluated experimentally. Comparative structure–activity analysis, molecular docking, molecular descriptor calculations, and in silico ADME profiling were additionally performed. Results: All compounds inhibited bovine pancreatic DNase I, with IC₅₀ values ranging from 34.42 ± 5.86 to 61.95 ± 9.61 μM. Compound 5a showed the highest inhibitory activity within the present series (IC₅₀ = 34.42 ± 5.86 μM). Comparison with five previously reported structurally related derivatives identified preliminary structure–activity trends, with 5a exhibiting the highest activity among the nine compounds considered. Molecular docking predicted a common preferred binding region within the major DNA-contact/catalytic region of DNase I and suggested possible noncovalent ligand–enzyme interactions. Molecular descriptor calculations revealed differences in the electronic properties of selected compounds, while in silico ADME analysis predicted generally favorable physicochemical and absorption-related properties together with potential pharmacokinetic liabilities. No measurable cytotoxicity was observed against the investigated cancer cell lines at the tested concentrations. Conclusions: Pyridine-containing squaramides represent a promising scaffold for further investigation as DNase I inhibitors, with 5a providing a useful starting point for subsequent structural optimization and mechanistic studies.

With the introduction of Navarixin in clinical trials, the role of squaric acid derivatives as bioisosteres gained popularity. Because of their distinctive electronic properties and hydrogen-bonding capacity, these compounds hold considerable promise for medicinal chemistry applications. In this study, a series of novel furan- and thiophene-containing squaric acid derivatives was synthesized via base-catalyzed nucleophilic substitution and characterized by spectroscopic techniques. The structures of three compounds were additionally confirmed by X-ray crystallography. Density functional theory calculations showed good agreement with the experimental vibrational spectra. In silico evaluation predicted favorable drug-like characteristics, including compliance with Lipinski’s rule of five and high gastrointestinal absorption. The cytotoxic activity of the synthesized compounds was assessed against HeLa, HT-29, HL-60, A-549, and MCF-7 cancer cell lines, as well as the non-cancerous CCL-1 cell line. Several derivatives displayed moderate to strong antiproliferative activity with selectivity toward malignant cells. Compound 3d exhibited the most pronounced improvement (five-fold) over Navarixin in HL-60 cells 5.81 µM, while compounds 3a and 3c demonstrated superior potency and selectivity in A-549 cells (10.33 µM and 9.65 µM). These findings identify squaric acid derivatives as promising candidates for further anticancer drug development and structure–activity relationship studies.