Synthesis and biological evaluation of new glutamic acid-based inhibitors of MurD ligase Tihomir Tomašic ´ a , Nace Zidar a , Veronika Rupnik a , Andreja Kovac ˇ a , Didier Blanot b , Stanislav Gobec a , Danijel Kikelj a , Lucija Peterlin Mašic ˇ a, * a University of Ljubljana, Faculty of Pharmacy, Aškerc ˇeva 7, 1000 Ljubljana, Slovenia b Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Univ Paris-Sud, 91405 Orsay, France article info Article history: Received 19 September 2008 Revised 28 October 2008 Accepted 29 October 2008 Available online 5 November 2008 Keywords: MurD ligase Glutamic acid Antibacterial agent Inhibitor abstract Mur ligases catalyze the biosynthesis of the UDP-MurNAc-pentapeptide precursor of peptidoglycan, an essential polymer of bacterial cell-wall. They constitute attractive targets for the development of novel antibacterial agents. Here we report on the synthesis of a series of 2,4-diaminoquinazolines, quinazo- line-2,4(1H,3H)-diones, 5-benzylidenerhodanines and 5-benzylidenethiazolidine-2,4-diones and their inhibitory activities against MurD from Escherichia coli. Compounds (R)-27 and (S)-27 showed inhibitory activity against MurD with IC 50 values of 174 and 206 lM, respectively, which makes them promising starting points for optimization. Ó 2008 Elsevier Ltd. All rights reserved. Bacterial resistance to currently available antibiotics has cre- ated an urgent need to discover novel effective antibacterial agents directed towards previously unexploited targets. 1,2 One of the best known and most validated targets for antibacterial therapy is the enzyme system responsible for peptidoglycan biosynthesis. 3 Pepti- doglycan is an essential cell-wall polymer whose main function is to provide the rigidity, flexibility and strength that are necessary for bacterial cells to grow and divide while withstanding high internal osmotic pressure. 4,5 As it is unique to prokaryotic cells, it represents an optimal target with respect to selective toxicity. A large number of antibiotics currently used in therapy act by inhib- iting late extracellular steps of peptidoglycan biosynthesis. In con- trast, the early intracellular steps of biosynthesis of cytoplasmic peptidoglycan precursor have been less exploited to date. 3,4 The biosynthesis of peptidoglycan is a complex two-stage process involving intracellular assembly of the UDP-MurNAc-pen- tapeptide which is subsequently translocated through the cyto- plasmic membrane to its outer side, where the polymerization reactions take place. 6–8 Enzymes of the Mur ligase family, MurC to MurF, catalyze a series of reactions leading to UDP-MurNAc- pentapeptide by stepwise addition of L-Ala (MurC), D-Glu (MurD), meso-diaminopimelic acid or L-Lys (MurE) and D-Ala-D-Ala (MurF) to the starting MurC substrate UDP-MurNAc. 3,6 The crystal structures of Mur ligases from different bacterial species are known and all reveal the same three-domain topology, with N-terminal and central domains responsible for binding of the UDP-precursor and ATP, respectively, while the C-terminal domain binds the condensing amino acid or dipeptide residue. 9–16 Surpris- ingly, sequence alignment of Mur ligase orthologues and para- logues revealed relatively low overall homologies, but importantly, quite high homology of residues present in the active sites. The ATP binding pocket in particular seems to be well con- served throughout the family. 17–20 The catalytic mechanism of Mur ligases has been studied in de- tail and they most probably operate by a similar chemical mecha- nism. Initially, the terminal carboxyl group of the UDP-substrate is activated by phosphorylation, resulting in formation of an acyl- phosphate intermediate which is subsequently attacked by the amino group of the condensing amino acid residue or dipeptide. The tetrahedral high-energy intermediate formed collapses with elimination of inorganic phosphate and concomitant peptide bond formation. 4,21 Moreover, based on the results of biochemical stud- ies of MurC and MurF, Mur ligases exhibit an ordered kinetic mech- anism in which ATP binds first to the free enzyme, followed by the corresponding UDP-substrate and condensing amino acid or dipep- tide last. 22,23 X-Ray experiments revealed two distinct ‘open’ conformations and one ‘closed’ conformation of MurD from Esche- richia coli, in which the binding of the substrates is accompanied by conformational closure of the C-terminal domain. 12,24 There have been several attempts to design MurC to MurF inhibitors by using substrate or transition-state analogues and structure-based design, reviewed by El Zoeiby et al. 3 and more recently by Barreteau et al. 6 and Štefanic ˇ Anderluh et al. 25 Recently, 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.10.129 * Corresponding author. Tel.: +386 (0)1 476 95 00; fax: +386 (0)1 425 80 31. E-mail address: lucija.peterlin@ffa.uni-lj.si (L.P. Mašic ˇ). Bioorganic & Medicinal Chemistry Letters 19 (2009) 153–157 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl