A series of inhibitors of the aspartate transcarbamoylase, an enzyme involved in pyrimidine nucleotide biosynthesis, has been synthesized. These inhibits are analogues of a highly potent inhibitor of this enzyme, N-phosphonacetyl-L-aspartate (PALA). Analogues have been synthesized with modifications at the α and β carboxylates as well as at the aspartate moiety. The ability of these compounds to inhibit the enzyme was evaluated. These studies, with functional group modified PALA derivatives, showed that amide groups can be a useful substitute of the carboxylate in order to reduced the charge on the molecule, and indicate that the relative position of the functional group in the β-position is more critical than the nature of the functional group. Some of the molecules synthesized here are potent inhibitors of the enzyme.Keywords allosteric enzyme; bi-substrate analog; aspartate carbamoyltransferase In mammals aspartate transcarbamoylase (ATCase) is a portion of a multifunctional enzyme (CAD) 1 which is required for de novo pyrimidine nucleotide biosynthesis. The ATCase portion of CAD catalyses the second step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate and L-aspartate to give N-carbamoyl-L-aspartate and inorganic phosphate. 2 ATCase has become a target for the development of anti-proliferative drugs and inhibitors of ATCase are considered as potential anti-tumor agents, since the levels of ATCase have been shown to be elevated in cancer cells. 3 N-(Phosphonoacetyl)-L-aspartate (PALA), a bi-substrate analogue of ATCase, 4 is a potent inhibitor which shows strong anti-proliferative 5, 6 and anti-tumor 7, 8 activities in cell culture. It has been examined in clinical trials as a possible anti-proliferation agent, however, a considerable drop in its effectiveness was observed. 9 This is probably due to the difficult translocation of PALA in to cells, 10 since the highly ionic nature of PALA makes it difficult for it to diffusion through the lipid bilayer of the cell membrane. 11No structural data is available for CAD or the ATCase portion of CAD, however the amino acid sequences of the mammalian and E. coli enzymes are 43% identical, and therefore the structure of the E. coli enzyme has often been used as a model for the structure of the ATCase portion of CAD. 12 All of the side chains important for catalytic activity in the E. coli enzyme * Corresponding author. Tel. +1 617 552 4558; e-mail: evan.kantrowitz@bc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Numerous analogues of PALA have been reported, unfortunately none of the...