ATP citrate lyase is an enzyme involved in mammalian lipogenesis and cholesterogenesis. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agents. Citric acid analogues 5-16 bearing electrophilic and latent electrophilic substituents were synthesized and evaluated as irreversible inhibitors of the enzyme. The design of these agents was based on the classical enzymatic mechanism where an active-site nucleophile (thiol) was believed to be critically involved in catalysis. Reversible inhibition (Ki's ranging from ca. 20 to 500 microM) was observed for compounds 5, 10, and 12-16. Compounds 6-9 and 11 had no appreciable affinity for enzyme (Ki > 1 mM). Time-dependent inactivation of the enzyme by 5-16 was not detected following long incubation times (> 1 h, 37 degrees C) at 2 mM inhibitor concentrations.