This work describes a search for hexokinase inhibitors based on the interactions analysis at the active site of the X-ray resolved o-tolulyl-glucosamine-hexokinase (OTG-HK) complex structure. As the actual enzyme sequence was unknown when the X-ray structure was made (only 30% homology), the structure of the complex was rebuilt by modelling on the X-ray structure frame which allowed residues in close vicinity to the inhibitor to be defined, particularly Glu249 and Gln278. Compounds with inhibitor-bearing groups able to interact with these residues were synthesized and assayed. Some of them revealed strong affinities, in the Km range for glucose. Kinetic analysis of their behaviour towards glucose and ATP together with spectroscopic studies using NMR, allowed the determination of the corresponding inhibition patterns and provided complementary information on HK.