Piperazine-thiophene hybrids as emerging bioactive scaffolds: synthesis, spectral characterization, in vitro evaluation and in silico insights.

Three piperazine-thiophene hybrid derivatives, namely quinolin-2-yl(4-(thiophene-2‑carbonyl)piperazin-1-yl)methanone (QTPM), (4-(4-nitrobenzoyl)piperazin-1-yl)(thiophen-2-yl)methanone (NPTM), and (4-picolinoylpiperazin-1-yl)(thiophen-2-yl)methanone (PPTM), were synthesized through a single-step amide-coupling strategy in moderate to good yields (60-70%). The synthesized compounds were characterized using 1H and 13C NMR spectroscopy, FT-IR spectroscopy, and ESI-MS/HRMS Mass, which collectively supported their proposed molecular structures. The in vitro biological activities of the synthesized derivatives were evaluated using heat-induced bovine serum albumin (BSA) denaturation and α-amylase inhibition assays. All three compounds exhibited concentration-dependent activity in both assays. PPTM showed the highest inhibition of BSA denaturation (82.08% at 400 μg/mL) and α-amylase activity (93.36% at 400 μg/mL), while QTPM also exhibited strong α-amylase inhibition (92.23%). Molecular docking studies against the selected biological targets revealed favorable interactions for all three derivatives, with QTPM showing the most favorable predicted binding affinities, including -6.88 kcal/mol against 1HNY and - 9.45 kcal/mol against 1PGG. Density Functional Theory (DFT) calculations demonstrated differences in frontier molecular orbital characteristics, with NPTM exhibiting the smallest HOMO-LUMO energy gap (3.8062 eV) and the highest electrophilicity index (6.3312 eV). Molecular electrostatic potential and Mulliken charge analyses identified the amide carbonyl oxygen and heteroaromatic nitrogen atoms as important electron-rich sites that may contribute to intermolecular interactions. In silico toxicity profiling indicated favorable predictions for several organ-specific toxicity endpoints; however, potential neurotoxicity and respiratory-toxicity alerts were identified, while NPTM additionally showed predicted carcinogenicity and mutagenicity alerts. Overall, the combined experimental and computational findings indicate that QTPM and PPTM represent promising lead structures within the piperazine-thiophene scaffold for further investigation as potential antidiabetic and anti-inflammatory agents. Further in vivo pharmacological, pharmacokinetic, and toxicological studies are required to validate their therapeutic potential and safety.