Synthesis and Biological Evaluation of Hydroxamate-Based Inhibitors of Glutamate Carboxypeptidase II Doris Stoermer, Qun Liu, Monicia R. Hall, Juliet M. Flanary, Ajit G. Thomas, Camilo Rojas, Barbara S. Slusher and Takashi Tsukamoto* Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA Received 25 February 2003; accepted 15 April 2003 Abstract—A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1 0 residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxy- carbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC 50 value of 220 nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure–activity relationships of GCP II inhibition. # 2003 Elsevier Science Ltd. All rights reserved. Glutamate carboxypeptidase II (GCP II, EC 3.4.17.21), also known as N-acetylated a-linked acidic dipeptidase (NAALADase) and prostate-specific membrane antigen (PSMA), is a metallopeptidase which cleaves N-acetyl- aspartylglutamate (NAAG) into N-acetylaspartate and glutamate in the nervous system. 1 This enzyme belongs to the M28 peptidase family comprised of co-catalytic metallopeptidases. 2 Peptidases in this family contain two metal ions forming a co-catalytic active site with ligands consisting of five amino acid residues. Inhibition of GCP II has gained considerable attention as a strat- egy to suppress glutamate excitotoxicity leading to neurological disorders including stroke, spinal cord injury, amyotrophic lateral sclerosis (ALS), peripheral neuropathy, chronic pain, schizophrenia, and epilepsy. 3 2-(Phosphonomethyl)pentanedioic acid (2-PMPA) 1a 4 is one of the first potent inhibitors of GCP II with a K i value of 0.2nM. 5 The high potency of compound 1a can be attributed to the strong chelation of the phosphonate group to an active site zinc atom as well as the interac- tion of the glutarate (pentanedioic acid) portion of the inhibitor with the glutamate recognition site of GCP II. 2-PMPA has been extensively utilized to study the mechanism and physiological role of GCP II as well as the potential therapeutic effects of GCP II inhibition. For example, we have demonstrated that 2-PMPA con- siderably reduces the ischemia-induced rise in extra- cellular glutamate and vigorously protects against injury in a neuronal culture model of ischemia and in rats after transient middle cerebral artery occlusion (MCAO). 6 We have extended our SAR studies to other zinc-bind- ing groups and identified phosphinate and thiol-based GCP II inhibitors 1b 7 and 2, 8 which exhibited in vivo efficacy in animal models of various neurological dis- orders. In order to further diversify the pharmacophore of GCP II inhibitors, we have examined hydroxamate group as an alternative zinc-binding group. The hydroxamate group of metalloprotease inhibitors is known to bind in a bidentate fashion to the active site zinc(II) ion and has proven to be one of the most effective zinc-binding 0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-894X(03)00407-4 Bioorganic & Medicinal Chemistry Letters 13 (2003) 2097–2100 *Corresponding author. Fax: +1-410-631-6797; e-mail: tsukamotot@ guilfordpharm.com