Unexpected Synthesis of a Furoxan Derivative from 3-Acetyl-2,4,6-Trimethylpyridine: Structural Characterization and Biological Evaluation

Herein, we report an unexpected pseudo-multicomponent transformation discovered during attempts to selectively nitrate the pyridine core of 3-acetyl-2,4,6-trimethylpyridine (3). Despite employing standard nitration conditions, including KNO3–H2SO4 and HNO3–H2SO4 mixtures, electrophilic substitution of the aromatic ring did not occur. Instead, the reaction sequence promoted an in situ nitrozation, dehydration to nitrile oxide intermediates, and subsequent [3+2]-cycloaddition involving two substrate molecules. This process yielded a novel, highly functionalized furoxan derivative, precisely identified as 3,4-bis(2,4,6-trimethylnicotinoyl)-1,2,5-oxadiazole 2-oxide (5). The molecular architecture of compound 5 was established by 1H and 13C NMR spectroscopy, mass spectrometry, elemental analysis, and single-crystal X-ray diffraction (XRD) analysis. To elucidate the stereochemical and electronic features governing compound 5, DFT calculations were performed at the ωB97X-D/6-311++G(d,p) level of theory. The experimental crystallographic disorder of the N-oxide oxygen atom was computationally rationalized by the thermodynamic near-degeneracy (ΔG < 0.63 kcal/mol) of two orientational isomers (5a and 5b). Furthermore, frontier molecular orbital analysis within the framework of perturbation theory accounted for the head-to-tail regioselectivity during cyclization, while wide energy gaps (ΔE = 8.13–8.27 eV) and high chemical hardness (η = 4.07–4.14 eV) underscored the kinetic stability of the heterocycle. Phenotypic and target-specific in silico profiling using PASS Online identified Matrix Metalloproteinase-9 (MMP-9) as a relevant target for potential hemorheological and cardioprotective applications. Validated molecular docking simulations across three human MMP-9 crystallographic domains (PDB: 8K5Y, 6ESM, 4XCT) demonstrated competitive binding affinities and balanced Ligand Efficiency metrics (LE = 0.26–0.29 kcal/mol/heavy atom), anchoring compound 5 within the catalytic pocket via conventional hydrogen bonds and π-mediated interactions. Finally, in vitro evaluations using a blood hyperviscosity model confirmed significant hemorheological efficacy, as compound 5 effectively prevented the rise in blood viscosity, outperforming the reference drug pentoxifylline. The convergence of computational insights and experimental functional activity establishes this novel bis(nicotinoyl)furoxan framework as a promising candidate for further hemorheological and cardioprotective applications.