30 Letters in Drug Design & Discovery, 2004, 1, 30-34 1570-1808/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd. Molecular Modeling, Synthesis And Biological Evaluation of Heterocyclic Hydroxamic Acids Designed as Helicobacter Pylori Urease Inhibitors E.M.F. Muri, H. Mishra, S.M. Stein and J.S. Williamson * Department of Medicinal Chemistry, University of Mississippi, University, MS 38677,USA Received August 29, 2003: Accepted October 21, 2003 Abstract: A computer-generated homology model of the antimicrobial target Helicobacter pylori urease was derived, using the x-ray crystal structure of Klebsiella aerogenes as a template, in order to design novel urease inhibitors. Based on these computational studies, several heterocyclic hydroxamic acid derivatives have been designed, synthesized, and examined for their ability to inhibit urease activity. Keywords: Helicobacter pylori, antimicrobial, urease, hydroxamic acids. INTRODUCTION Almost 90% of the human population is believed infected with the spiral-shaped, Gram-negative bacterium Helicobacter pylori (H. pylori), making it one of the most infectious agents known [1]. After numerous studies on the pathogenic role of this bacterium, it is now widely accepted that H. pylori is a major causative factor in peptic ulcer diseases [2]. Antibiotic eradication therapies in the clinic have been shown to heal gastritis, provide a cure for many patients suffering from peptic ulcers, and even generate a remission of MALT carcinomas. While the majority of H. pylori infections can be controlled by currently available antibiotic therapies [3], antibiotic resistant strains of the microorganism are becoming frequent [4]. Infection of the human gastrointestinal tract, specifically the mucous lining of the stomach, by H. pylori requires that the microorganism neutralize or adapt to the otherwise lethally acidic environment. H. pylori utilizes its own metabolism of urea to ammonia by the enzyme urease to counteract the harsh acidic acid environment of the stomach. By analogy, inhibition of this urea to ammonia catalysis should halt this vital neutralization mechanism [5]. Molecules designed to specifically target and inhibit the H. pylori urease offer the potential of effective and selective chemotherapies. In general, ureases are inhibited by a variety of agents including fluorides [6], thiols [7,8] and hydroxamic acids [9,10]. Several mono-amino acid and dipetide derivatives containing hydroxamic acid moieties have been synthesized and found to be potent and specific inhibitors of H. pylori urease [11]. In order to explore the structural parameters associated with these hydroxamic acid inhibitors, we developed a homology model using the urease crystal structure from Klebsiella aerogenes [12] (EC 3.3.1.5) as a template, which we have reported earlier [13]. Through the utilization of computational design we have now synthesized a series of heterocyclic hydroxamic acid derivatives. In addition, we have made an initial determination of the effect of these derivatives on urease activity using jack bean urease inhibitor assay. *Address correspondence to this author at the Department of Medicinal Chemistry, University of Mississippi, University MS 38677, USA; Email: mcjsw@olemiss.edu RESULTS AND DISCUSSION Acetohydroxamic acid was docked into the active pocket of this homology-modeled urease and the most probable configuration of the enzyme-inhibitor complex was assessed by molecular dynamics studies. Comparative Molecular Field Analysis (CoMFA) was then carried out on a variety of dipeptide hydroxamic acid derivatives. Our CoMFA model included 24 dipeptide hydroxamic acid derivatives, using the conformation of structural ligands based on the acetohydroxamic-enzyme complex as obtained through homology modeling, docking and finally molecular dynamics. The predictive value of the model was evaluated and verified using a test set that was not included in the original model development. Overlapping the contour maps derived from the CoMFA model with the amino acids associated with the enzyme active pocket resulted in a model that provide an initial conceptualization and understanding of the steric and electrostatic requirements for ligand binding to and inhibition of H. pylori urease [13]. Figures 1-2 illustrate the alignment of dipeptide hydroxamic acid dataset as well as the electrostatic and steric maps used in the design of our heterocyclic hydroxamic acid derivatives [13]. Based on these results, we designed and synthesized a series of heterocyclic hydroxamic acid derivatives. Possessing the required pharmacophore groups necessary to increase selective urease binding stability in addition to binding selectivity, as a hydroxamic acid and ionizable amine moieties, the structural design was directed at the synthesis of potential hydroxamic acid inhibitors that are likely to exhibit increased biological stability as compared with the dipeptide and amino acid derivatives [14]. Scheme 1 shows the preparation of the imidazole hydroxamic acid derivatives 7a-b, where the commercially available imidazole (1) was initially nitrated to yield the 4(5)-nitro-1H -imidazole (2 ) [15]. Alkylation of 2 in the presence of base has been reported to yield predominantly the 1,4-disubstituted isomer [16]. The esters 3a-b were obtained by alkylation with the respective alkylating agent in the presence of K 2 CO 3 and TBAI as a phase-transfer catalyst [17]. The next step was the catalytic reduction of the nitro-ester compounds 3a-b yielding the unstable 4-amino- imidazoles 4a-b, which were directly protected with a tert-