Design, Synthesis, and Evaluation of Novel Organophosphorus Inhibitors of Bacterial Ureases Stamatia Vassiliou, ‡ Agnieszka Grabowiecka, † Paulina Kosikowska, † Athanasios Yiotakis, ‡ Pawel Kafarski, † and Lukasz Berlicki †, * Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw UniVersity of Technology, Wyb. Wyspian ´skiego 27, 50-370 Wroclaw, Poland, Laboratory of Organic Chemistry, Department of Chemistry, UniVersity of Athens, Panepistimopolis, Zografou, 15771 Athens, Greece ReceiVed May 15, 2008 A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P- methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in Vitro against urease from Bacillus pasteurii and Proteus Vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with K i ) 170 nM (against B. pasteurii enzyme) for the most active N-(N′-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid. Introduction Urease (urea amidohydrolase, E.C. 3.5.1.5) is an enzyme that catalyzes hydrolysis of urea to ammonia and carbamate, which is the final step of nitrogen metabolism in living organisms. 1,2 Carbamate decomposes rapidly and spontaneously, yielding a second molecule of ammonia. These reactions cause significant increase of solution pH. Bacterial ureases are large heteropolymeric metalloproteins with nickel(II) ions present in their active sites. 3-6 A significant amino acid sequence similarity was observed between all ureases of a bacterial origin. 7,8 The mechanism of enzymatic reaction has been studied extensively by several research groups for many years. 2,5,9-11 On the basis of several crystal structures of complexes of Bacillus pasteurii urease, Ciurli and co-workers proposed the most reliable enzymatic reaction mechanism. 10 The active site of the native enzyme binds three water molecules and a hydroxide ion bridged between two nickel ions. Urea replaces these three water molecules and is bound by a network of hydrogen bonds as well as by the nickel ions. 12 An activated carbon atom of urea is attacked by the Ni-bridging hydroxide ion, forming a tetrahedral transition state. Subsequently, am- monia is released from the active site followed by the negatively charged carbamate. Several classes of compounds are known to show considerable inhibitory activity against this enzyme with phosphoramidates being the most active. 13-17 It was shown that phenyl phosphor- diamidate (PPD a ) and its 4-substituted derivatives exhibit inhibitory properties in nanomolar range (PPD was a competitive slow-binding inhibitor with K i * ) 0.6 nM against B. pasteurii enzyme). 16,18 Its mode of action relays on hydrolysis in the active site and release of phosphorodiamidic acid (1), which is the actual enzyme inhibitor and represents enzymatic reaction transition state analogue. 10,15 However, hydroxamic acids are the best recognized urease inhibitors. 13,19-22 The simplest analogue in this group is acetohydroxamic acid, which exhibited a competitive slow-binding inhibition with K i ) 2.6 µM against Klebsiella aerogenes enzyme. Various hydroxamate analogues, several aminoalkanehydroxamic acids, their N-substituted de- rivatives, as well as dipeptides, were examined (N-glycylgly- cinehydroxamic acid exhibited IC 50 ) 0.79 µM against Heli- cobacter pylori urease). 23,24 Interestingly, simple organosulfur compounds represented, for example, by -mercaptoethanol, also exhibited considerable inhibitory activity. 25 This phenomenon was explained by the analysis of the crystal structure of -mercaptoethanol-urease complex, in which the sulfur atom was bridged between the two nickel ions present in the active site. 6 The studies on novel urease inhibitors are essential not only for the basic research on urease biochemistry but also for the possible development of a highly needed therapy for urease mediated bacterial infections. 1,8,26,27 Ammonia released in the urease catalyzed reaction is either a direct cause of clinical conditions or a crucial factor to the pathogen survival and a host colonization. Ureolytic activity of several microorganisms, i.e., Proteus mirabilis, Proteus Vulgaris, and Ureaplasma urealyticum, is involved in the formation of urinary tract stones, which may lead to the chronic inflammation of kidney and its pelvis. 28-31 Additionally, urinary catheter obstruction in patients is caused by its colonization by urease-producing microorgan- isms, mainly P. mirabilis. 32,33 Moreover, the overproduction of ammonia by infectious microorganisms may contribute to ammonia encephalopathy or hepatic coma. 34-36 Acetohydroxamic acid, a potent urease inhibitor, was shown to be an efficient drug against diseases caused by ureolytic bacteria. 37-39 Another mechanism of urease involvement in pathogenic bacteria infection relies on the creation of a microenvironment suitable for the pathogen existence. Helicobacter pylori infection of stomach is possible only after local neutralization of gastric acid by released ammonia. 27,40,41 Furthermore, high concentration of ammonia disturbs mucosal permeability, in particular hy- drogen ions passage through mucosal surface, and causes formation of peptic ulcers. 42,43 The immense significance of this medical problem was emphasized by the 2005 Nobel Prize in * To whom correspondence should be addressed. Phone: +48 71 320 40 80. Fax: +48 71 328 40 64. E-mail: lukasz.berlicki@pwr.wroc.pl. † Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology. ‡ Laboratory of Organic Chemistry, Department of Organic Chemistry, University of Athens. a Abbreviations: PPD, phenyl phosphordiamidate; Cbz, benzyloxycar- bonyl, TMS, trimethylsilyl; EDC 1-ethyl-3-(3-dimethylaminopropyl) car- bodiimide; HOBt, Hydroxybenzotriazol; DIPEA, diisopropylethylamine. J. Med. Chem. 2008, 51, 5736–5744 5736 10.1021/jm800570q CCC: $40.75  2008 American Chemical Society Published on Web 08/22/2008