[structure: see text] The synthesis of N-acylsulfonamide 6, which is an analogue of beta-aspartyl-AMP, is described. This compound appears to be the first and only potent inhibitor of human asparagine synthetase that has been described to date. The N-acylsulfonamide 6 exhibits slow-onset inhibition kinetics, with a K(i) of 728 nM. Preparation and characterization of two additional N-acylsulfonamide analogues has also demonstrated the importance of hydrogen-bonding interactions in the recognition of the AS inhibitor with the enzyme. These observations provide the basis for the discovery of new compounds with application in the treatment of drug-resistant leukemia.