J. Med. Chem. 2009, 52, 4095–4098 4095 DOI: 10.1021/jm900504c
*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; email,
[email protected]. a Abbreviations: Aβ, amyloid-β; AChE, acetylcholinesterase; AD, Alzheimer's disease; APP, amyloid precursor protein; BuChE, butyrylcholinesterase; IRE, iron-responsive element; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; NFT, neurofibrillary tangles; PAS, peripheral anionic site; ROS, reactive oxygen species; CNS, central nervous system.
clinical use. Long-term use of strong chelators with poor target specificity is expected to interact with beneficial biometals and affects the normal physiological functions of essential metal-requiring metalloenzymes, thereby promoting undesirable side effects. To overcome some of these limitations, recently, several new chelators or prochelators with improved target specificity as potential drug candidates for AD have been developed in our and others’ laboratories.7-10 Because of the complex etiology of AD and the involvement of different but related dysfunctions in its progression, development of new multifunctional drugs for treating AD has increasingly attracted interest in recent years.3,11-13 We have developed a number of multifunctional chelators with the neuroprotective and neurorestorative propargylamine moiety14,15 for AD therapy; these new multifunctional chelators have good permeability into both the brain and normal cells with poor target specificity.3,15-23 Here we report on a novel prochelator strategy as an approach to develop a novel class of multifunctional prochelators with enhanced target specificity.24 These novel prochelators are designed to inhibit AChE with a concurrent release in the brain of multifunctional metal chelators possessing the neuroprotective and neurorestorative propargylamine moiety.14,15 AChE inhibitor moiety was chosen, since AChE inhibitors have symptomatic anti-AD activity in the clinic. Furthermore, AChE inhibitors have also been shown to reduce the amyloid burden in cellular and transgenic model of AD with cognitive improvement.25,26 These novel prochelators that we have designed would have little affinity for metal ions to avoid interfering with healthy metal metabolism, a common toxicity associated with chelation therapies. However, as prodrugs, they would lose their masks while binding and inhibiting AChE as pseudoinhibitors in the brain, releasing active chelators that passivate excess cerebral metal ions and protect neuronal cells against ROS. Since metal chelators and AChE inhibitors may have diseasemodifying effects by modulating NFT and/or reducing Aβ deposition and since AChE inhibitors are the most successful drugs for the symptomatic treatment of AD, these novel prochelators hold great promise for combating the causes and the symptoms of AD. To design such novel prochelators, we started from our newly developed bifunctional chelator 5-(4-propargylpiperazin-1-ylmethyl)-8-hydroxyquinol 1 (HLA20)3,17,20-23 and marketed AChE inhibitors rivastigmine and donepezil.25 Recent studies have shown that AChE inhibitors binding to the active site and the peripheral anionic site (PAS) have the potential to inhibit the Aβ aggregation induced by AChE.27,28 Rivastigmine has three moieties (carbamyl, phenyl, and ethylmethylamino moieties) binding to the active site of AChE, while donepezil possesses a pharmacophoric moiety [(5,6-dimethoxy-1-indanon-2-yl)methylpiperidine] interacting with the PAS and the middle of AChE gorge.29,30 We therefore incorporated and merged these moieties into the structure of 1, in which the carbamyl moiety also acts as a protective group to mask the quinolinol oxygen, a key atom for chelating metal ions (class A in Chart 1). To test the feasibility of our novel prochelator strategy, we synthesized our first-generation prochelator 5-(4-propargylpiperazin-1-ylmethyl)-8-hydroxyquinolinyldimethylcarbamate
r 2009 American Chemical Society
Published on Web 06/01/2009
Site-Activated Multifunctional Chelator with Acetylcholinesterase and NeuroprotectiveNeurorestorative Moieties for Alzheimer’s Therapy Hailin Zheng,†,§ Moussa B. H. Youdim,*,‡ and Mati Fridkin*,† † Department of Organic Chemistry, the Weizmann Institute of Science, Rehovot 76100, Israel, and ‡Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases and Department of Pharmacology, Technion-Rappaport Family Faculty of Medicine Haifa, 31096, Israel. § Current address: Intra-Cellular Therapies, Inc., 3960 Broadway, New York, NY 10032
Received April 21, 2009 Abstract: A novel strategy to develop site-activated multifunctional chelators for targeting multiple etiologies of Alzheimer’s disease is reported. The novel prochelator HLA20A with improved cytotoxicity shows little affinity for metal ions until it is activated by binding and inhibiting acetylcholinesterase (AChE), releasing an active chelator HLA20 that modulates amyloid precursor protein (APP) regulation and β-amyloid (Aβ) reduction, suppresses oxidative stress, and passivates excess metal ions (Fe, Cu, and Zn) in the brain.
Studies have indicated that cerebral biometals (Fe, Cu, and Zn) dyshomeostasis and oxidative stress in the brain are closely associated with the formation of β-amyloid (Aβa) plaques and neurofibrillary tangles (NFT), the hallmarks in the brain of Alzheimer’s disease (AD) patients.1,2 The abnormally high levels of Fe and Cu in affected areas of the brain catalyze the formation of reactive oxygen species (ROS), which further aggravates oxidative stress contributing to τ hyperphosphorylation and NFT formation.1-3 Iron increases the production of amyloid precursor protein (APP) translation via activation of APP mRNA iron-responsive element (IRE) and consequently Aβ formation.4,5 Dyshomeostasis of the biometals and their interactions with Aβ cause Aβ aggregation and deposition.1-5 Metal chelators have the ability to attenuate the broad spectrum of oxidative stress associated neuropathologies, as well as APP translation, Aβ generation, and amyloid plaques and NFT formation.2,3,6 These effects have rendered metal chelators as very promising diseasemodifying drugs for Alzheimer’s disease. The metal chelators under investigation as potential drugs for AD include desferrioxamine and clioquinol, which are not target (brain) specific metal chelators with significant drawbacks with respect to bioavailability and/or cytotoxicity.7-9 The poor target specificity and/or brain permeability and consequential clinical safety of these metal chelators have limited their widespread
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Chart 1. Design Strategy Leading to Prochelator-AChE Inhibitors (Class A) by Incorporating the Structural Features of Rivastigmine and Donepezil into a Multifunctional Metal Chelator 1
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.
Figure 1. (A) Time-dependent inhibition of AChE in rat brain homogenate 2 and rivastigmine at 1 μM. (B) Concentration-dependent inhibition of AChE and BuAChE in rat brain homogenate by HLA20A inhibitions was determined by a modified Ellman method after preincubations of the enzymes with 2 for 3 h, using acetylthiocholine as a substrate for AChE and butyrylthiocholine as a substrate for BuAChE. Data are mean values ( SEM of three to five independent experiments each done in triplicate. The IC50 values were calculated using a nonlinear curve.
Scheme 1. Synthesis of the New Prochelator 2a
a Reagents 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.
2 (HLA20A).24 As shown in Scheme 1, 2 was obtained by the carbamylation of 1 with dimethylcarbamyl chloride in the presence of sodium hydride. 1 was synthesized as previously described.20 It is expected that 2 would have little to no affinity for metal ions but inhibit AChE with a concomitant release in the brain of 1. 1 is a neuroprotective chelator with capabilities of modulating APP regulation and Aβ reduction, suppressing oxidative stress, and inhibiting Aβ aggregation induced by metal ions (Cu and Zn).3,17,20-23
To determine the in vitro activity of 2 against AChE and BuChE, a modified Ellman’s method (see Supporting Information) was employed using rivastigmine as a reference. Experiments revealed that 2 inhibited AChE activity in a time-dependent manner with slightly more potency than rivastigmine (Figure 1A). Figure 1B shows ChE inhibition curves for 2. The best fitting curves had IC50 of 0.50 ( 0.06 μM and 42.58 ( 6.67 μM for AChE and BuChE, respectively. The IC50 values suggest that 2 is a potent AChE inhibitor with high selectivity toward AChE (IC50(BuChE)/IC50(AChE) ≈ 85). As BuChE may also play an important role in the metabolism of ACh in the central nervous system (CNS), some inhibition of BuChE may be beneficial in treating AD.31 However, the selectivity toward AChE found in 2 may be advantageous in term of target specificity and potential side effects; since AChE is mainly located in the CNS and BuChE is more abundant in the peripheral system, the weak inhibition of BuChE not only would help avoid the potential side effects of nonselective ChE inhibitors such as tacrine32 but also would help minimize the possibility of AChE-mediated cleavage of 2 to a strong metal chelator 1 before entering the CNS, thus improving its target specificity. To investigate whether 2 can chelate metal ions (Fe, Cu, Zn), spectrophotometric studies were conducted. As shown in curves b and c of Figure 2, addition of either CuSO4 or ZnCl2 to a solution of 2 did not result in any significant changes in the 200-700 nm absorption spectra, suggesting little or no complex formation between 2 with Cu2+ or Zn2+. Addition of FeCl3 or FeSO4 to a solution of 2 produced a slight increase at 300 nm, which may indicate a weak interaction (Figure 2 d,e). The absence of new features suggests that tight Fe complexes did not form. To show the possibility of enzymatic removal of the carbamyl moiety in 2, we used an AChE-mediated hydrolysis model. Carbamate AChE inhibitors such as rivastigmine have been shown to be readily hydrolyzed by AChE with a concomitant release in the brain of the OH metabolite.29 As expected, AChE was found to readily cleave carbamyl moiety in 2 and release the OH metabolite 1, as revealed by thin-layer chromatography and UV-visible spectroscopy (Figure 3). As shown in Figure 3, introduction of FeSO4 to an extract from a solution of AChE-activated 2 led to the emergence of three new bands at about 340, 450, and 575 nm matching that of the 1-Fe complex as reported previously;20,23 a new band around 375 mn appeared upon the addition of CuSO4 to the extract, demonstrating the formation of the 1-Cu complex. These results suggest that 2 was hydrolyzed to 1 by AChE.
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metal (Fe, Cu, and Zn) dyshomeostasis in the brain. It can therefore 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 Weizmann Institute for generous support of this work. Supporting Information Available: Details of syntheses and biology tests. This material is available free of charge via the Internet at http://pubs.acs.org.
References
Figure 3. Prochelator 2 inhibits AChE with a concomitant release of a multifunctional chelator 1 that sequesters metal ions involved in the etiology of AD [UV/vis absorbance spectra in 5% NH4Ac (pH 7)]: (a) 2 (0.2 mM); (b) 1 (0.2 mM); (c and d) 2 was activated prior to addition of CuSO4 (0.2 mM) and FeSO4 (0.2 mM), respectively.
Figure 4. Effects of 1 and 2 on cell viability in human SH-SY5Y neuroblastoma cells. Data are mean values ( SEM of three independent experiments.
This finding was reinforced by mass spectrometry analysis of the extract, which gave m/z 282.28, corresponding to m/z of [1 + H]+. To examine the cytotoxicity effects of 2 and 1, human SHSY5Y neuroblastoma cells were exposed to the test compounds for 48 h and the cell viability was tested by the 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium (MTT) assays. As reported in Figure 4, 2 and 1 did not show modified cell viability at 1 μM. 1 induced a decrease of cell viability at 10 μM (60%), 25 μM (87%), and 50 μM (98%). By contrast, prochelator is nontoxic to the cells at 10 μM, and even at high concentrations (25 or 50 μM) it is much less toxic than 1. In summary, our results demonstrate the feasibility of a novel prochelator strategy for AD therapy in which the incorporation of the pharmacophores from rivastigmine into 1 produces a novel prochelator 2. 2 shows much improved cytotoxicity compared to 1 and has little to no affinity for metal ions until it inhibits AChE and BuChE with a concomitant release of a multifunctional chelator 1. This novel strategy has advantages over the current prochelator strategy, since it will be able not only to minimize the potential toxicity associated with nonspecific chelators but also to offer multiple activities against AChE, Aβ aggregation, oxidative stress, and
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