A series of acidic amino acids has been prepared and evaluated in an effort to identify the structural features required for binding to and inhibiting the high affinity uptake system that clears L-glutamate from the synaptic cleft during excitatory amino acid-mediated neurotransmission in the mammalian CNS. L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS. This excitatory amino acid (EAA) or closely related ones, such as L-aspartate or L-homocysteate, mediate fast synaptic transmission at AMPA and KA receptors as well as participate in higher order processes coupled to Ca++ (NMDA receptor) and phosphoinositide (ACPD receptor) signaling. 1 In addition to its role in normal neuronal communication, glutamate acts as a potent neurotoxin when its extracellular concentrations reach excessive levels. Glutamate-mediated neuronal injury, referred to as excitotoxicity, is believed to contribute to CNS pathology in a wide spectrum of disorders, including ischemia, hypoglycemia, epilepsy, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease.2 This dichotomy clearly suggests that levels of glutamate must be carefully maintained at concentrations sufficient to mediate excitatory transmission, yet not so high as to induce excitotoxic-initiated pathology. Specific high affinity transporters, which are integral membrane proteins present on presynaptic terminals and the astrocytes surrounding the synapse, catalyze the translocation of glutamate and related acidic amino acids across the lipid bilayer of the plasma membrane and play a key role in maintaining this balance. Their ability to rapidly clear EAA agonists from the synaptic cleft is thought to be a critical step in terminating the excitatory signal, recycling the transmitter, and maintaining the extracellular concentration of glutamate below that which could induce excitotoxic injury.3 This crucial role has become evident as the potential pathological consequences of reduced function have been investigated. For example, the apparent inverse relationship between excitotoxic injury and transport capacity is consistent with the observation that much larger amounts of L-glutamate are required to produce lesions in vivo than of EAA agonists (e.g., kainate) that are not efficiently transported.4 Similarly, Roberts and colleagues have reported that the neurotoxic action of β-threo-OH-aspartate (β-THA), a competitive inhibitor of glutamate uptake, results from its exacerbation of the excitotoxic action of glutamate.5 In vitro studies with cortical cultures have demonstrated that reductions in transport capacity, produced by either decreasing astrocyte densities or attenuating transport directly, dramatically increase neuronal sensitivity to glutamate-mediated damage.6be Furthermore, data is now emerging that ties the pathological consequences of compromised transport to specific neurodegenerative diseases. Thus, Palmer et al.7 have reported a decrease in glutamate transport sites in Alzheimer's disease on the basis of reduced substrate binding (i.e., 3H-D-aspartate), while Rothstein et al. have reported a marked reduction in the maximal velocity of glutamate uptake in synaptosomes prepared from the spinal cords of patients with amyotrophic lateral sclerosis (ALS).8 Considerable evidence suggests that EAA transport in the CNS is not mediated by a single system, but by a number of distinct transporters that have been difficult to fully differentiate owing to a lack of specific substrates and inhibitors. Pharmacological and kinetic studies have discerned variations as a function of transporter location (neuronal vs. glial, forebrain vs. cerebellar, and synaptosomes vs. synaptic vesicles), as well as ion-dependence (sodium vs. chloride). Of the various systems, the high-affinity sodium-dependent glutamate transporter is the most thoroughly characterized in terms of its pharmacology, mechanism, and distribution.3c9 The significance of distinguishing and characterizing these systems goes well beyond interesting biochemical differences and is central to: (i) evaluating their roles in excitatory physiology and pathology. (ii) accurately identifying changes that accompany age or diseases, and (iii) assessing the consequences of reduced function. Toward this goal, we have attempted to prepare pharmacologically specific analogues of L-glutamate with which to probe transport biochemistry and function. As has been the case for the EAA receptors, pharmacological characterization of the EAA transporters is closely tied to their prevalence and to the availability of pharmacologically selective ligands. Thus, previous work has focused primarily on the sodium-dependent transporter, defining its basic specificity by quantifying the ability of a wide range of EAA analogues to reduce the accumulation of radiolabeled substrates (e.g., 3H-L-glutamate and 3H-D-aspartate). The transport inhibitors generally share the common feature of being α-amino acids with a second acidic group separated from the α-COOH by 2-4 methylene groups. 3c,9a Structure-activity studies indicate that the distal OH group can be derivatized to a hydroxamate, as in L-aspartate-β-hydroxamate, or replaced by a sulfonate group, as in cysteic acid. Some modification of the carbon backbone is also tolerated: β-THA and dihydrokainate (DHK) are well known competitive inhibitors. Interestingly, while the transporter shows a strong preference for binding L-glutamate over the D-enantiomer, both L- and D-aspartate are excellent substrates. Despite the usefulness of these compounds in the initial characterization of the uptake systems, their lack of selectivity is a serious drawback. For each there are several energetically accessible conformations, which allows binding at sites other than the transporter. As a result, these compounds cannot inhibit glutamate uptake without also activating one (or more) of the EAA receptors. Similarly, such compounds would be expected to be of little value in distinguishing the subtle differences that may exist among the EAA transport systems. Improved selectivity would thus constitute an important advance in this area, and one way to achieve this objective would be the use of analogs with more rigid structures resembling the conformations of glutamate required for binding to the transporter(s) but not to the excitatory receptors. 1* Recently this approach has led to the identification of several interesting transport inhibitors, including L-α-(carboxycyclopropyl)glycine derivatives (L-CCG-III, Figure 6):11 and cis- 1-aminocyclobutane- 1,3-dicarboxylate (CACB, Figure 6). 12 Several years ago we began a systematic study of azacyclic dicarboxylate conformer mimics and their interactions with the EAA receptors and transporters. In this report we describe structure-activity data for all twelve isomers of pyrrolidine 2,3-dicarboxylate (2,3-PDC), pyrrolidine 2,4-dicarboxylate (2,4-PDC), and azetidine 2,3-dicarboxylate (2,3-ADC), as well as several more highly substituted derivatives of 2,4-PDC, at the sodium-dependent transporter in synaptosomes prepared from rat forebrain.