Caitlyn M. Vortolomei

Courtney A. Walsh

Ingo Janser

Ranjith K. Meka

Romy F.J. Janser

Comptes Rendus Chimie

FuEuInCoa Db D1.coWura Almuraracfrowin[7reC. R. Chimie 16 (2013) 660–664A ArtReAcAvKea,bEthInhPeUr*Un16httll paper/Mémoirethacrynic acid as a lead structure for the development of potentrease inhibitorsgo Janser a,*, Caitlyn M. Vortolomei b, Ranjith K. Meka b,urtney A. Walsh b, Romy F.J. Janser bepartment of Chemistry, Eastern Michigan University, 48197 Ypsilanti, MI, USAepartment of Chemistry, New Mexico Institute of Mining and Technology, 87801 Socorro, NM, USA IntroductionUrease (EC 3.5.1.5) is an enzyme that catalyzes thenversion of urea into ammonia and carbon dioxide [1,2].hile urea amidolyase breaks down urea in two steps,ease does this in a single step [3]. Urease can be found ingreat variety of algae, bacteria, plants, and fungi [1,4,5].though, the structure, type and number of subunits, theirolecular weight and the amino acid sequence of theeases differ, the amino acid sequence of the active sitese conserved and therefore, the mechanism of the enzymetivity is the same for all ureases [5,6]. Urease isolatedm the plant jack bean is a homohexameric moleculehose active site contains two nickel ions that are involved binding of substrates as well as of some of the inhibitors,8]. Each jack bean urease subunit possesses 15 cysteinesidues, which makes urease a thiol-rich enzyme [6,9].However, without denaturation of the enzyme, only six ofthe 15 cysteine residues are accessible for reagents. One ofthese residues, cysteine-592, is recognized to play a criticalrole in the catalytic activity of the enzyme [10,11]. Ureasesare ubiquitous in nature and it is known that they aredirectly associated with the formation of infection stonesand contribute to the pathogenesis of several infectiousdiseases like e.g. pyelonephritis, an ascending urinary tractinfection [12]. Additionally, they are an important factor inthe pathogenesis of many clinical conditions [13,14].Helicobacter pylori (H. pylori) produces high quantities ofurease and thereby large amounts of ammonia are formedwhich makes it possible for the bacteria to inhabit thestomach [12]. This high amount of ammonia in thestomach is now accepted as the major cause of pepticulcers [2,15]. For this reason, urease inhibitors haveattracted a lot of attention as potent anti-ulcer drugs[16]. Due to the diverse function of the enzyme, the studyof urease inhibition not only has medical but alsoenvironmental and agronomic significance [6]. The devel-opment of potent and specific inhibitors could lead to thetreatment of infections caused by urease-producingR T I C L E I N F Oicle history:ceived 9 December 2012cepted after revision 28 March 2013ailable online 6 May 2013ywords:-unsaturated ketonesacrynic acidibitorptic ulcereaseA B S T R A C TEthacrynic acid and a series of its analogues were synthesized and subsequently evaluatedfor their inhibitory effect on jack bean urease. Ethacrynic acid showed, even at lowconcentrations, very potent inhibitory activity against the enzyme. For ethacrynic acid, theinhibition potential increased with increasing preincubation time of ethacrynic acid andenzyme, whereas for some other compounds a higher preincubation time lead to asignificant reduction of their activity. We could demonstrate that the a,b-unsaturatedcarbonyl unit of our compounds is mandatory to inhibit the enzyme, possibly due to itsability to bind to cysteine residues in the active site of the jack bean urease. 2013 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved. Corresponding author. Department of Chemistry, Eastern Michiganiversity, 541 Science Complex, 48197 Ypsilanti, MI, USA.E-mail address: ijanser@emich.edu (I. Janser).Contents lists available at SciVerse ScienceDirectComptes Rendus Chimiew ww.s c ien ced i rec t . c o m31-0748/$ – see front matter  2013 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.p://dx.doi.org/10.1016/j.crci.2013.03.020btifokc(I[1aboceacpsina22aasaI-FcI. Janser et al. / C. R. Chimie 16 (2013) 660–664 661acteria [17]. In 2003, Kawase et al. reported for the firstme that several a,b-unsaturated ketones are inhibitorsr jack bean urease. The most potent a,b-unsaturatedetones were cyclic and of low-molecular weight, e.g. 2-yloheptene-1-one (IC50 = 0.16 mM), 2-cyclohexene-1-oneC50 = 0.69 mM), 2-cyclopentene-1-one (IC50 = 0.97 mM)2].To the best of our knowledge, no further report on other,b-unsaturated carbonyl compounds as potential inhi-itors of urease has been published. Ethacrynic acid, a loopr high ceiling diuretic, possesses an a,b-unsaturatedarbonyl unit and is used to treat high blood pressure anddema [18,19]. We have recently synthesized severalnalogues of ethacrynic acid lacking the a,b-unsaturatedarbonyl unit and discovered that some of our analoguesossess a significant potency to inhibit the migration ofeveral cancer cell lines [20,21]. We herein report thehibitory effect of ethacrynic acid (IV-1) and seven of itsnalogues (IV-2-8) (Fig. 1) on jack bean urease.. Experimental.1. General procedure for the preparation of ethacrynic acidnd its analoguesThe synthesis of ethacrynic acid (IV-1) and its sevennalogues (IV-2-8, Fig. 1) was accomplished by a three-tep reaction shown in Scheme 1. The Friedel–Craftscylation reaction of the phenol or the substituted phenols1-4 (Scheme 1), respectively, with propanoyl chloride(n = 1) or butanoyl chloride (n = 2), respectively, wasperformed in the presence of powdered aluminiumchloride (AlCl3) in carbon disulfide, as recently describedby us [22]. Compounds II-1-8 were purified by flashcolumn chromatography on silica gel and consecutivelyrefluxed in acetone for 48 hours in the presence of 1.2equivalents of ethyl bromoacetate and two equivalentspotassium carbonate (K2CO3) to yield compounds III-1-8.In the third step, an aldol condensation reaction, com-pounds III-1-8 were refluxed in an ethanol/water (50/50)mixture for 24 hours in the presence of two equivalents offormaldehyde and 2.5 equivalents of K2CO3. CompoundsIV-1-8 were obtained by flash chromatography using ahexanes/ethyl acetate/methanol mixture as the eluent.2.2. Urease inhibition assayThe assay mixture containing 50 mL jack bean urease(5 mg of type III urease dissolved in 0.5 mL phosphatebuffered saline [PBS]) and 1 mL of the test compound(dissolved in dimethyl sulfoxide [DMSO]) was preincu-bated for 60, 120, 180, and 240 minutes at roomtemperature in a 96-well assay plate. After preincubation,150 mL urea broth containing phenol red pH indicator(9.68 g urea medium dissolved in 250 mL reverse osmosis[RO] water) was added to each well. Once the substrate andthe indicator were added, a micro plate reader was used tomeasure the initial absorbance for each well at 595 nm.The results (change in absorbance per min) were processedusing SoftMax Pro software (Molecular Devices). Inhibitionig. 1. Ethacrynic acid (IV-1) and seven of its analogues (IV-2-8). Compounds IV-1 and IV-2, IV-3 and IV-4, IV-5 and IV-6, as well as IV-7 and IV-8 areonsidered as chain length analogues.Scheme 1. Synthesis of ethacrynic acid (IV-1) and seven of its analogues (IV-2-8).pe(Ovaco0.4pe3.unagefcourprfoshbetivfoac(ICetit 0.(ICchtra1.foThbyofhiIVcoCo(0(ICtiocohy(ICTaInhanCIIIIIIIIHAaI. Janser et al. / C. R. Chimie 16 (2013) 660–664662rcentages were calculated using the formula 100–Dtestwell/ODcontrol)  100. In order to determine IC50lues, inhibition studies were carried out at variousncentrations of the test compound (1.0 mM, 0.5 mM, mM, 0.3 mM, 0.2 mM, and 0.1 mM). All reactions wererformed in triplicate. Results and discussionCompounds IV-1-8 (Fig. 1) all of which possess an a,b-saturated carbonyl unit were subsequently testedainst jack bean urease. We initially tested the inhibitoryfects of our eight compounds (IV-1-8) at variousncentrations thereby keeping the concentration ofease constant. Before adding the urea broth, weeincubated the enzyme with the respective compoundr 60 minutes. The results of our enzyme assays areown in Table 1. Ethacrynic acid (IV-1) itself inhibits jackan urease by 28% (0.5 mM) or 43% (1.0 mM), respec-ely (IC50 = 1.21 mM). Similar observations were mader its chain length analogue (IV-2) whose inhibitorytivity of 26% (0.5 mM) or 41% (1.0 mM), respectively50 = 1.32 mM), is slightly lower than that observed forhacrynic acid (IV-1). For compound IV-3, we found thatinhibits jack bean urease by 50% at a concentration of5 mM and even by 65% at a concentration of 1.0 mM50 = 0.50 mM). Similar results were obtained for itsain length analogue IV-4 (48% inhibition at a concen-tion of 0.5 mM and 63% inhibition at a concentration of0 mM). Even higher inhibitory activities could be foundr compound IV-5 and its chain length analogue IV-6.ese compounds are capable of inhibiting the enzyme 64% or 65%, respectively, at the lower concentration 0.5 mM and by 78% or 79%, respectively, at thegher concentration of 1.0 mM (IV-5, IC50 = 0.15 mM;-6, IC50 = 0.13 mM). Similar results were found formpounds IV-7 and its chain length analogue IV-8.mpound IV-7 inhibited jack bean urease by 62%.5 mM) and by 76% at a concentration of 1.0 mM50 = 0.18 mM) and compound IV-8 showed 61% inhibi-n at a concentration of 0.5 mM and 75% inhibition at ancentration of 1.0 mM (IC50 = 0.21 mM). As a control,droxyurea (IC50 = 0.21 mM) and acetohydroxamic acid50 = 0.05 mM), both known inhibitors of jack beanurease, were used. After an incubation time of 1 hour, theinhibitory activity of the most potent compounds (IV-5-8)was equal or better than that of hydroxyurea. However,they are three to four times less active than acetohy-droxamic acid. An early conclusion is that the chain lengthof the acyl group does not seem to have a significant effecton the inhibitory activity of the corresponding compound,whereas the substitution pattern at the aromatic ring ofthe ethacrynic acid seems to have a great influence on theactivity of the corresponding compound (chloro- versushydrogen- versus methoxy substituent).We were now interested if an increase in preincubationtime has an effect on the inhibition of the enzyme.Therefore, we investigated the inhibitory activities of ourcompounds at various preincubation times (1, 2, 3, and4 hours). Much to our delight, the inhibitory activities ofethacrynic acid (IV-1) and its chain length analogue (IV-2)were significantly increased at higher incubation times.The highest activity observed was after a preincubation of4 hours. After this preincubation time, ethacrynic acid (IV-1) inhibited the enzyme by 71% at a concentration of1.0 mM (IC50 = 0.25 mM). Surprisingly, with increasingpreincubation time, the activities of the other compounds(IV-5-8) did not increase as significantly as for ethacrynicacid and its chain length analogue after an incubation timeof 4 hours (IV-5, IC50 = 0.08 mM; IV-6, IC50 = 0.05 mM, IV-7,IC50 = 0.07 mM; IV-8, IC50 = 0.10 mM) (Table 1). Theobservation we made for compounds IV-3 and IV-4 wascompletely unexpected. The inhibitory activity of thesecompounds decreased from 48% inhibition (0.5 mM, 1 hourpreincubation) to nearly no observable activity after apreincubation of 4 hours. We speculated that compoundsIV-3 and IV-4 were degraded by the enzyme, which wasaffirmed by the fact that we were able to detect thepresence of compounds IV-3 and IV-4 in the assay mixtureafter the preincubation of 1 hour, but not after 4 hours ofincubation. The resulting product of the degradationcouldn’t be identified. As a control, after a preincubationof 4 hours, we added more of compound IV-3 and IV-4 andcould observe a returning inhibitory activity. Why thisdecomposition happens for compounds IV-3 and IV-4 andnot for the chloro- or methoxy substituted compounds isnot understood yet. It is noteworthy that all of the testedcompounds inhibit the urease reversibly. Adding moreurea to the assay mixture resulted in further activity of theenzyme.In another experiment, we wanted to determine, if theaddition of sulfhydryl compounds, such as benzyl mer-captan or cyclohexyl mercaptan, reduces the inhibitoryactivity of our compounds or even prevents the inhibitionof jack bean urease. Sulfhydryl compounds are Michaeldonors and can attack the b-carbon of the a,b-unsaturatedcarbonyl unit, thereby preventing its reaction withcysteine residues in the active site of the jack bean ureaseThe results from our enzyme assays show that oneequivalent of the corresponding sulfhydryl compound(benzyl mercaptan or cyclohexyl mercaptan) affected theinhibitory activity of ethacrynic acid IV-1 and its analoguesIV-3, IV-5, IV-7 significantly. We preincubated the enzymewith the respective compounds IV-1, IV-3, IV-5, and IV-7 atvarious concentrations in the presence of one equivalent ofble 1ibition of jack bean urease by ethacrynic acid (IV-1) and seven of itsalogues (IV-2-8) at different incubation times.ompound IC50 (mM)1 ha 2 ha 3 ha 4 haV-1 (Ethacrynic acid) 1.21 0.51 0.32 0.25V-2 1.32 0.55 0.36 0.28V-3 0.50 0.81 2.21 V-4 0.48 0.89 2.25 V-5 0.15 0.12 0.10 0.08V-6 0.13 0.11 0.09 0.05V-7 0.18 0.16 0.10 0.07V-8 0.21 0.20 0.17 0.10ydroxyurea 0.21 0.20 0.21 0.22cetohydroxamic acid 0.05 0.04 0.04 0.05Preincubation time.thta4coaaocohthinninth4attitina(ec(ctpCTEthdI. Janser et al. / C. R. Chimie 16 (2013) 660–664 663e corresponding sulfhydryl compound (benzyl mercap-n or cyclohexyl mercaptan, respectively) for 1 and for hours and found that the inhibitory activity of theompounds was reduced by approximately half of theirriginal activity (Table 2). As expected, the inhibitoryctivity of our control (acetohydroxamic acid) was notffected by the additives, no loss of activity could bebserved. This is an indication that the a,b-unsaturatedarbonyl unit is responsible for the inhibitory activity ofur compounds described above. In order to verify ourypothesis, we tested all eight precursors (III-1-8), lackinge a,b-unsaturated carbonyl unit (Scheme 1) for theirhibitory activity on jack bean urease. As expected,one of the tested compounds were able to measurablyhibit the urease, not even at concentrations higheran 1.0 mM.. ConclusionIn summary, we have tested ethacrynic acid (IV-1)nd seven of its analogues (IV-2-8) for their potencyo inhibit jack bean urease. We could demonstrate thathe inhibitory activities of ethacrynic acid (IV-1) ands chain length analogue (IV-2) were significantlycreased with higher incubation times. The highestctivity observed was after a preincubation of 4 hoursIC50 = 0.25 mM or IC50 = 0.28 mM, respectively). How-ver, the highest inhibitory activity was found forompound IV-6 (IC50 = 0.05 mM), compound IV-7IC50 = 0.07 mM), compound IV-5 (IC50 = 0.08 mM), andompound IV-8 (IC50 = 0.10 mM) after a preincubationime of 4 hours. It is noteworthy that all four compoundsossess a methoxy group at the aromatic system.ompounds IV-3 (IC50 = 0.50 mM, 1 h preincubation)and IV-4 (IC50 = 0.48 mM, 1 h preincubation) are alsocapable of inhibiting jack bean urease. However, theirinhibitory activity is reduced with increasing incubationtime. It can be speculated that these compounds aredegraded by the enzyme. Further investigations toaccount for these findings are necessary. Due to thedistinct inhibitory activities of compounds IV-5, IV-6, IV-7, and IV-8, it can be speculated that the substituentsattached to the aromatic system (chloro- versus hydro-gen- versus methoxy substituent) have a great influenceon the activity of the corresponding compound. Apossible explanation for these distinct inhibitory activ-ities is that methoxy groups favor the Michael additionreaction due to the fact that they activate the aromaticsystem through a resonance effect. On the other hand, thechloro substituents deactivate the ring through anelectron-withdrawing inductive effect and thereby pos-sibly disfavoring the Michael addition reaction. Furtherstructure–activity relationship experiments have to beperformed in order to get a better understanding of theinfluence of the substituents. The addition of sulfhydrylcompounds reduces the activity of ethacrynic acid and itsanalogues significantly. This is insofar not surprisingsince the sulfur of the sulfhydryl compound can attackthe b-carbon of the a,b-unsaturated carbonyl unit,thereby preventing its reaction with cysteine residuesin the active site of the jack bean urease. Testingprecursors of our compounds which lack the a,b-unsaturated carbonyl unit (III-1-8) for their inhibitoryactivity on urease reveals the importance of the a,b-unsaturated carbonyl unit because none of them show ameasurable inhibition of the enzyme. Further investiga-tions to design even more potent ethacrynic acidanalogues for the inhibition of jack bean urease arecurrently underway in our laboratory.AcknowledgementsThe financial support of the US National Institutes ofHealth (P20 RR016480) under the INBRE program of theNational Center for Research Resources (NCRR) is greatlyappreciated.References[1] H.L.T. Mobley, R.P. Hausinger, Microbiol. Rev. 53 (1989) 85.[2] H.L.T. Mobley, M.D. Island, R.P. Hausinger, Microbiol. Rev. 59 (1995)451.[3] D.H.M.L.P. Navarathna, S.D. Harris, D.D. Roberts, K.W. Nickerson, FEMSYeast Res. 10 (2010) 209.[4] A. Sirko, R. Brodzik, Acta Biochim. Pol. 47 (2000) 1189.[5] B. Krajewska, J. Mol. Catal. B: Enzym. 59 (2009) 22.[6] T. Tanaka, M. Kawase, S. 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Chem. 25 (2010) 296.able 2ffects of additives on the inhibitory activity of ethacrynic acid (IV-1) andree of its analogues (IV-3, IV-5, and IV-7) against jack bean urease atifferent incubation times.Compound Additives IC50(mM)1 ha 4 haIV-1(Ethacrynic acid)None 1.21 0.25Benzyl mercaptan (1 equiv) 2.65 0.50Cyclohexyl mercaptan (1 equiv) 2.48 0.55IV-3 None 0.50 Benzyl mercaptan (1 equiv) 1.15 Cyclohexyl mercaptan (1 equiv) 1.11IV-5 None 0.15 0.08Benzyl mercaptane (1 equiv) 0.35 0.20Cyclohexyl mercaptan (1 equiv) 0.28 1.17IV-7 None 0.18 0.07Benzyl mercaptane (1 equiv) 0.35 0.15Cyclohexyl mercaptan (1 equiv) 0.34 0.16AcetohydroxamicacidNone 0.05 0.01Benzylmercaptane (1 equiv) 0.05 0.01Cyclohexyl mercaptan (1 equiv) 0.05 0.01a Preincubation time.[15[16[17[18[19I. Janser et al. / C. R. Chimie 16 (2013) 660–664664] Z. Amtul, R.A. Atta-ur-Rahman, M.I. Siddiqui, Choudhary, Curr. Med.Chem. 9 (2002) 1323.] V.U. Ahmad, J. Hussain, H. Hussain, A.R. Jassbi, F. Uullah, M.A. Lodhi, A.Yasin, M.I. 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Ethacrynic acid as a lead structure for the development of potent urease inhibitors

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Ethacrynic acid as a lead structure for the development of potent urease inhibitors

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