Exploration of Novel Pyrazole Derivatives: Design, Synthesis, and Integrated Biological Profiling (In Vitro and In Silico) of Their Antioxidant, Antibacterial, and Antimitotic Properties.

This study investigates the biological activities of synthetic derivatives 7a-b, focusing on antioxidant, antibacterial, antimitotic, and mitotic index assays after NMR and mass spectrometry characterization. Derivative 7b showed superior antioxidant activity, with 75.19 ± 0.11% inhibition at 2.5 µmol/mL and an IC of 0.85 ± 0.02 µmol/mL, outperforming 7a (IC = 1.44 ± 0.13 µmol/mL). In antibacterial tests, 7b inhibited Staphylococcus aureus (16.3 mm zone), while 7a showed limited effects, particularly against Gram-negative bacteria. For antimitotic activity, 7b inhibited 60% of seed germination, while 7a had no effect. The mitotic index revealed that 7b inhibited up to 80% at 20 µmol/mL, similar to colchicine, whereas 7a had minimal impact. Overall, 7b demonstrated strong antioxidant, antibacterial, and antimitotic potential, while 7a showed lower activity across all tests. On the in-silico level, compound 7a demonstrated strong binding affinities across three biological targets. Against the antioxidant target Kelch-Neh2, it achieved a docking score of -9.08 kcal/mol and formed interactions with 14 residues, including Thr560. For antibacterial activity, it outperformed standard drugs with a docking score of -6.77 kcal/mol and hydrogen bonding to DNA gyrase residues Gly77 and Asn46. In anti-mitotic studies, it showed a docking score of -7.19 kcal/mol, forming stable interactions with the tubulin binding pocket, including Asn258. On the same compound, ADME analysis reveals high absorption and moderate interaction with P-glycoprotein. Toxicity predictions suggest a non-carcinogenic, non-mutagenic, non-cytotoxic, and non-immunotoxic profile. Density Functional Theory (DFT) indicated favorable electronic properties, including a low energy gap (0.2011 eV), moderate dipole moment, and high softness, supporting good bioactivity and stability. Molecular dynamics simulations confirmed structural stability of all three complexes over 100 ns, with RMSD values within 1.2-2.4 Å. MM-GBSA binding free energy analyses further supported stable binding, with ΔGbind values remaining strongly negative across simulation time frames. PCA and PDF analyses demonstrated consistent conformational stability for compound 7a compared to reference compounds, while Free Energy Landscape plots indicated stable and energetically favorable conformational states.