This study focused on Salla disease and cancer metastasis. Nine sialyltransferase inhibitors (ST01–ST09) were designed and synthesized, and their structures and purities were confirmed by 1H NMR, 13C NMR, and mass spectrometry. Enzyme inhibition assays demonstrated that ST04 and ST09 exhibited significant inhibitory activity against sialyltransferases ST6GAL1 and ST3GAL1, with ST09 showing an IC₅₀ value of 3.80 μM, superior to the reported compounds S1047 and S1069. Molecular docking simulations further revealed that the phenyl moiety in ST04 and ST09 can form stable hydrophobic interactions and π–π stacking within the ST6GAL1 active pocket, explaining their strong inhibitory activity. AutoDock4 calculations indicated that binding energy and Ki serve as key predictive indicators of inhibitory potential. In cell-based experiments, ST06 and ST09 effectively reduced sialic acid levels in HEK293 SLC17A5 knockout cells, restoring them to near-normal levels, thereby demonstrating therapeutic potential at the cellular level. Overall, ST04 and ST09 exhibited the best performance in enzyme assays, ST06 and ST09 showed notable effects in cell assays, and ST09 consistently demonstrated strong activity across multiple evaluation levels, highlighting it as a promising candidate for further development as a sialyltransferase inhibitor.