Abstract
Compound 8, (5Z)-2-hydroxy-4-methyl-6-oxo-5-[(5-phenylfuran-2-yl)methylidene]-5,6-dihydropyridine-3-carbonitrile, is an effective NEK6 kinase inhibitor with demonstrated anticancer and neuroprotective activity. However, its poor aqueous solubility (3 μg/mL) presents a significant barrier to therapeutic development. To address this limitation, we developed a graphene-based nanocarrier system by conjugating compound 8 and its fluorinated derivatives (8-F and 8-CF(3)) onto structurally uniform few-layer graphene nanoparticles (GNPs) obtained via ball-milling and liquid-phase exfoliation (B60). The resulting conjugates (8@B60, 8-F@B60, and 8-CF(3)@B60) were thoroughly characterized by UV-vis, IR, and Raman spectroscopy, as well as by TEM and STEM-EDX analysis. Spectroscopic and elemental data confirmed effective drug loading and structural preservation of the B60 nanocarriers. Drug release experiments further confirmed thermally triggered desorption of 8 from the GNPs surface in aqueous conditions, highlighting the potential of B60-based conjugates as controlled release systems. A key finding of this work is the reversible hydration of compound 8 in aqueous solution, resulting in a colorless, nonconjugated species. Detailed spectroscopic and computational studies revealed that this hydrated form likely represents the dominant species under physiological conditions. Crucially, molecular docking and molecular dynamics simulations demonstrated that hydration does not compromise the binding affinity of compound 8 for the NEK6 active site. These insights provide a molecular-level rationale for the design and evaluation of drug delivery systems based on nanographene platforms in aqueous environments.