pubs.acs.org/jmcPublished on Web 06/01/2009r 2009 American Chemical Society

J. Med. Chem. 2009, 52, 4095–4098 4095

DOI: 10.1021/jm900504c

Site-Activated Multifunctional Chelator with

Acetylcholinesterase and Neuroprotective-Neurorestorative Moieties for Alzheimer’s

Therapy

Hailin Zheng,†,§ Moussa B. H. Youdim,*,‡ andMati Fridkin*,†

†Department of Organic Chemistry, the Weizmann Institute ofScience, Rehovot 76100, Israel, and ‡Eve Topf and USA National

Parkinson Foundation Centers of Excellence for NeurodegenerativeDiseases and Department of Pharmacology, Technion-Rappaport

Family Faculty of Medicine Haifa, 31096, Israel. §Current address:Intra-Cellular Therapies, Inc., 3960 Broadway, NewYork, NY 10032

Received April 21, 2009

Abstract: Anovel strategy to develop site-activatedmultifunctionalchelators for targeting multiple etiologies of Alzheimer’s disease isreported. The novel prochelator HLA20A with improved cytotoxi-city shows little affinity for metal ions until it is activated by bindingand inhibiting acetylcholinesterase (AChE), releasing an activechelator HLA20 that modulates amyloid precursor protein (APP)regulation and β-amyloid (Aβ) reduction, suppresses oxidativestress, and passivates excessmetal ions (Fe, Cu, andZn) in the brain.

Studies have indicated that cerebral biometals (Fe, Cu, andZn) dyshomeostasis and oxidative stress in the brain areclosely associated with the formation of β-amyloid (Aβa)plaques and neurofibrillary tangles (NFT), the hallmarks inthe brain of Alzheimer’s disease (AD) patients.1,2 The abnor-mally high levels of Fe and Cu in affected areas of the braincatalyze the formation of reactive oxygen species (ROS),which further aggravates oxidative stress contributing to τhyperphosphorylation andNFT formation.1-3 Iron increasesthe production of amyloid precursor protein (APP) transla-tion via activation of APP mRNA iron-responsive element(IRE) and consequently Aβ formation.4,5 Dyshomeostasis ofthe biometals and their interactions with Aβ cause Aβ aggre-gation and deposition.1-5 Metal chelators have the ability toattenuate the broad spectrum of oxidative stress associatedneuropathologies, as well as APP translation, Aβ generation,and amyloid plaques and NFT formation.2,3,6 These effectshave rendered metal chelators as very promising disease-modifying drugs for Alzheimer’s disease. The metal chelatorsunder investigation as potential drugs for AD include desfer-rioxamine and clioquinol, which are not target (brain) specificmetal chelators with significant drawbacks with respect tobioavailability and/or cytotoxicity.7-9 The poor target speci-ficity and/or brain permeability and consequential clinicalsafety of these metal chelators have limited their widespread

clinical use. Long-term use of strong chelators with poortarget specificity is expected to interact with beneficial biome-tals and affects the normal physiological functions of essentialmetal-requiring metalloenzymes, thereby promoting undesir-able side effects. To overcome some of these limitations,recently, several new chelators or prochelators with improvedtarget specificity as potential drug candidates for AD havebeen developed in our and others’ laboratories.7-10

Because of the complex etiology ofADand the involvementof different but related dysfunctions in its progression, devel-opment of new multifunctional drugs for treating ADhas increasingly attracted interest in recent years.3,11-13 Wehave developed a number of multifunctional chelators withthe neuroprotective and neurorestorative propargylaminemoiety14,15 for AD therapy; these new multifunctional chela-tors have good permeability into both the brain and normalcells with poor target specificity.3,15-23 Here we report on anovel prochelator strategy as an approach to develop a novelclass of multifunctional prochelators with enhanced targetspecificity.24 These novel prochelators are designed to inhibitAChE with a concurrent release in the brain of multifunc-tional metal chelators possessing the neuroprotective andneurorestorative propargylaminemoiety.14,15AChE inhibitormoiety was chosen, since AChE inhibitors have symptomaticanti-AD activity in the clinic. Furthermore, AChE inhibitorshave also been shown to reduce the amyloid burden in cellularand transgenicmodel ofADwith cognitive improvement.25,26

These novel prochelators that we have designed would havelittle affinity for metal ions to avoid interfering with healthymetal metabolism, a common toxicity associated with chela-tion therapies. However, as prodrugs, they would lose theirmaskswhile bindingand inhibitingAChEaspseudoinhibitorsin the brain, releasing active chelators that passivate excesscerebral metal ions and protect neuronal cells against ROS.Since metal chelators and AChE inhibitors may have disease-modifying effects by modulating NFT and/or reducing Aβdeposition and since AChE inhibitors are the most successfuldrugs for the symptomatic treatment of AD, these novelprochelators hold great promise for combating the causesand the symptoms of AD.

To design such novel prochelators, we started fromour newlydeveloped bifunctional chelator 5-(4-propargylpiperazin-1-yl-methyl)-8-hydroxyquinol 1 (HLA20)3,17,20-23 and marketedAChE inhibitors rivastigmine and donepezil.25 Recent studieshave shown that AChE inhibitors binding to the active site andthe peripheral anionic site (PAS) have the potential to inhibitthe Aβ aggregation induced by AChE.27,28 Rivastigmine hasthree moieties (carbamyl, phenyl, and ethylmethylamino moi-eties) binding to the active site of AChE, while donepezilpossesses a pharmacophoric moiety [(5,6-dimethoxy-1-inda-non-2-yl)methylpiperidine] interacting with the PAS and themiddle of AChE gorge.29,30 We therefore incorporated andmerged these moieties into the structure of 1, in which thecarbamyl moiety also acts as a protective group to mask thequinolinoloxygen,akeyatomfor chelatingmetal ions (classA inChart 1).

To test the feasibility of our novel prochelator strategy,we synthesized our first-generation prochelator 5-(4-propargyl-piperazin-1-ylmethyl)-8-hydroxyquinolinyldimethylcarbamate

*To whom correspondence should be addressed. For M.B.H.Y.:phone, 972-4-8295-290; fax, 972-4-8513-145; e-mail, youdim@tx.technion.ac.il. For M.F.: phone, 972-8-934 2505; fax, 972-8-934 4142; e-mail, mati.fridkin@weizmann.ac.il.

aAbbreviations: Aβ, amyloid-β; AChE, acetylcholinesterase; AD, Alz-heimer's disease; APP, amyloid precursor protein; BuChE, butyrylcholines-terase; IRE, iron-responsive element; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFT, neurofibrillary tangles; PAS, periph-eral anionic site; ROS, reactive oxygen species; CNS, central nervous system.

4096 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

2 (HLA20A).24 As shown in Scheme 1, 2 was obtained by thecarbamylation of 1 with dimethylcarbamyl chloride in thepresence of sodium hydride. 1 was synthesized as previouslydescribed.20 It is expected that 2would have little to no affinityfor metal ions but inhibit AChE with a concomitant release inthe brainof1. 1 is a neuroprotective chelatorwith capabilities ofmodulating APP regulation and Aβ reduction, suppressingoxidative stress, and inhibiting Aβ aggregation induced bymetal ions (Cu and Zn).3,17,20-23

To determine the in vitro activity of 2 against AChE andBuChE, a modified Ellman’s method (see Supporting Infor-mation) was employed using rivastigmine as a reference.Experiments revealed that 2 inhibited AChE activity in atime-dependent manner with slightly more potency thanrivastigmine (Figure 1A). Figure 1B shows ChE inhibitioncurves for2. Thebest fitting curves had IC50 of 0.50( 0.06μMand 42.58( 6.67 μMforAChE andBuChE, respectively. TheIC50 values suggest that2 is a potentAChE inhibitorwithhighselectivity toward AChE (IC50(BuChE)/IC50(AChE) ≈ 85).As BuChEmay also play an important role in themetabolismof ACh in the central nervous system (CNS), some inhibitionof BuChE may be beneficial in treating AD.31 However, theselectivity toward AChE found in 2 may be advantageous interm of target specificity and potential side effects; sinceAChE is mainly located in the CNS and BuChE is moreabundant in the peripheral system, the weak inhibition ofBuChE not only would help avoid the potential side effects ofnonselective ChE inhibitors such as tacrine32 but also wouldhelpminimize the possibility of AChE-mediated cleavage of 2to a strong metal chelator 1 before entering the CNS, thusimproving its target specificity.

To investigate whether 2 can chelate metal ions (Fe, Cu,Zn), spectrophotometric studies were conducted. As shown incurves b and c of Figure 2, addition of either CuSO4 or ZnCl2to a solutionof 2didnot result in any significant changes in the200-700 nm absorption spectra, suggesting little or no com-plex formation between 2 with Cu2+ or Zn2+. Addition ofFeCl3 or FeSO4 to a solution of 2 produced a slight increase at300 nm, whichmay indicate a weak interaction (Figure 2 d,e).The absence of new features suggests that tight Fe complexesdid not form.

To show the possibility of enzymatic removal of the carba-myl moiety in 2, we used an AChE-mediated hydrolysismodel. CarbamateAChE inhibitors such as rivastigmine havebeen shown to be readily hydrolyzed by AChE with aconcomitant release in the brain of the OH metabolite.29 Asexpected, AChEwas found to readily cleave carbamyl moietyin 2 and release the OHmetabolite 1, as revealed by thin-layerchromatography andUV-visible spectroscopy (Figure 3). Asshown in Figure 3, introduction of FeSO4 to an extract from asolution of AChE-activated 2 led to the emergence of threenew bands at about 340, 450, and 575 nm matching that ofthe 1-Fe complex as reported previously;20,23 a new bandaround 375 mn appeared upon the addition of CuSO4 to theextract, demonstrating the formation of the 1-Cu complex.These results suggest that 2 was hydrolyzed to 1 by AChE.

Chart 1. Design Strategy Leading to Prochelator-AChE In-hibitors (Class A) by Incorporating the Structural Features ofRivastigmine and Donepezil into a Multifunctional MetalChelator 1

Figure 1. (A) Time-dependent inhibition of AChE in rat brainhomogenate 2 and rivastigmine at 1 μM. (B) Concentration-depen-dent inhibition of AChE and BuAChE in rat brain homogenate byHLA20A inhibitions was determined by amodified Ellmanmethodafter preincubations of the enzymes with 2 for 3 h, using acetylthio-choline as a substrate for AChE and butyrylthiocholine as asubstrate for BuAChE. Data are mean values ( SEM of three tofive independent experiments each done in triplicate. The IC50

values were calculated using a nonlinear curve.

Scheme 1. Synthesis of the New Prochelator 2a

aReagents and conditions: (i) NaH (1.2 equiv), THF, 0 �C, 0.5 h;

(ii) (CH3)2NCOCl (1 equiv), THF, 0 �C to room temp overnight.

Figure 2. UV/vis absorption spectra of 2 (0.2 mM in 5% NH4Ac,pH 7) in the absence and presence of metal salts (0.2 mM): (a) 2; (b)2 + CuSO4; (c) 2 + ZnCl2; (d) 2 + FeSO4; (e) 2 + FeCl3.

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This finding was reinforced by mass spectrometry analysisof the extract, which gavem/z 282.28, corresponding tom/z of[1+ H]+.

To examine the cytotoxicity effects of 2 and 1, human SH-SY5Y neuroblastoma cells were exposed to the test com-pounds for 48 h and the cell viability was tested by the3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium (MTT)assays. As reported in Figure 4, 2 and 1 did not showmodifiedcell viability at 1 μM. 1 induced a decrease of cell viability at10 μM (60%), 25 μM (87%), and 50 μM (98%). By contrast,prochelator is nontoxic to the cells at 10 μM, and even at highconcentrations (25 or 50 μM) it is much less toxic than 1.

In summary, our results demonstrate the feasibility of anovel prochelator strategy for AD therapy in which theincorporation of the pharmacophores from rivastigmine into1 produces a novel prochelator 2. 2 shows much improvedcytotoxicity compared to 1 and has little to no affinity formetal ions until it inhibits AChE and BuChE with a con-comitant release of a multifunctional chelator 1. This novelstrategy has advantages over the current prochelator strategy,since it will be able not only to minimize the potential toxicityassociatedwithnonspecific chelators but also to offermultipleactivities against AChE,Aβ aggregation, oxidative stress, and

metal (Fe, Cu, and Zn) dyshomeostasis in the brain. It cantherefore be considered a novel avenue for AD therapy.

Acknowledgment. We thank the Alzheimer Association(U.S.), Alzheimer Drug Discovery Foundation (New York),Technion-Research and Development, and the WeizmannInstitute for generous support of this work.

Supporting Information Available: Details of syntheses andbiology tests. This material is available free of charge via theInternet at http://pubs.acs.org.

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Figure 3. Prochelator 2 inhibits AChE with a concomitant releaseof amultifunctional chelator 1 that sequestersmetal ions involved inthe etiology of AD [UV/vis absorbance spectra in 5% NH4Ac (pH7)]: (a) 2 (0.2 mM); (b) 1 (0.2 mM); (c and d) 2was activated prior toaddition of CuSO4 (0.2 mM) and FeSO4 (0.2 mM), respectively.

Figure 4. Effects of 1 and 2 on cell viability in human SH-SY5Yneuroblastoma cells. Data are mean values ( SEM of three in-dependent experiments.

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