molecules Communication
Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer’s Disease Mike Jones 1 , Jun Wang 1 , Shona Harmon 1 , Beata Kling 2 , Jörg Heilmann 2 and John F. Gilmer 1, * 1 2
*
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland;
[email protected] (M.J.);
[email protected] (J.W.);
[email protected] (S.H.) Institute of Pharmacy, University of Regensburg, 93053 Regensburg, Germany;
[email protected] (B.K.);
[email protected] (J.H.) Correspondence:
[email protected]; Tel.: +353-1-896-2795
Academic Editors: Michael Decker and Diego Muñoz-Torrero Received: 2 March 2016; Accepted: 23 March 2016; Published: 1 April 2016
Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective Abstract: butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 µM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 µM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ˘ 0.020 Trolox Equivalent. Keywords: neuroprotection; Alzheimer’s disease; antioxidant; cholinesterase inhibitor; hybrid
1. Introduction Alzheimer’s disease (AD) is now the most common neurodegenerative disease, with more than 20 million cases worldwide [1]. AD is characterised by global cognitive decline and associated neuropathological findings which may include neuronal loss, neurofibrillary tangles, neuritic plaques and amyloid angiopathy. Cholinergic loss is the single most replicated neurotransmitter deficiency in AD, and AChE has been a drug target for the treatment of AD since the emergence of this cholinergic hypothesis over 30 years ago [2]. This followed recognition that the cognitive impairments in AD correlated with cholinergic deficits such as reduced synaptic acetylcholine synthesis and choline acetyltransferase (ChAT) activity. The introduction of the cholinesterase inhibitors (ChEIs)—first tacrine then donepezil, rivastigmine, and galantamine—has made an important contribution to the management and well-being of early stage AD patients. Selective inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) e.g., donepezil and unselective inhibitors (ChEIs) e.g., rivastigmine, induce dose-limiting adverse effects such as bradycardia, nausea, and diarrhea [3,4]. There are challenges facing this field in identifying new agents that have improved efficacy or side effect profile. Medicinal chemistry themes that can be recognised as a response to this include a switch in focus from AChE to butyrylcholinesterase (BuChE; EC 3.1.1.8) [5–7]. This is because
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of the observations that BuChE assumes a more important role in the AD brain during disease progression Another major theme has been thethedesign of hybrids able toprogression modulate observations [8–10]. that BuChE assumes a more important role in AD brain during disease simultaneously aspectshas of the complex Alzheimer pathobiology [11,12]. simultaneously There is a lot of current [8–10]. Anotherseveral major theme been the design of hybrids able to modulate several interest, forthe example, the designpathobiology of hybrid compounds able is to ainhibit cholinesterase aspects of complexinAlzheimer [11,12]. There lot of current interest,and for attenuate example, neuronal apoptosis associated with tissue damage reactive oxygen species [13]. The antioxidant in the design of hybrid compounds able to inhibitand cholinesterase and attenuate neuronal apoptosis system loseswith effectiveness duringand the aging process and species oxidative damage has been observed associated tissue damage reactive oxygen [13]. The antioxidant systembefore loses the formationduring of AD-specific β-amyloid plaques is an effectiveness the aging pathological process and oxidative damage has[14]. beenα-Lipoic observedacid before theinteresting formation substance in this context because it plays an essential role in mitochondrial dehydrogenase reactions. of AD-specific pathological β-amyloid plaques [14]. α-Lipoic acid is an interesting substance in this Lipoate, or its reduced dihydrolipoate, reacts with reactive oxygen species such Lipoate, as superoxide context because it playsform, an essential role in mitochondrial dehydrogenase reactions. or its radicals, radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen [15]. α-Lipoate reduced hydroxyl form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, is taken up and reduced in cellsacid, and peroxyl tissues to dihydrolipoate. is also exported to hydroxyl radicals, hypochlorous radicals, and singletDihydrolipoate oxygen [15]. α-Lipoate is taken up and extracellular reduced in cells and tissues to dihydrolipoate. is also exported toand theextracellular extracellular the medium; therefore, protection isDihydrolipoate afforded to both intracellular medium; therefore, is afforded both intracellular and Thus, environments. Thus,protection α-lipoate would seemtoan ideal substance in theextracellular treatment ofenvironments. oxidative brain and α-lipoate would involving seem an ideal substance in the treatment of has oxidative brainbeen and linked neural to disorders neural disorders free radical processes. Lipoic acid previously the ChI involving free radicaland processes. Lipoic acid producing has previously been linked to the ChI compounds tacrine compounds tacrine quinazolinimines potential bimodal antioxidant cholinesterase and quinazolinimines potential antioxidant inhibitory therapeutics inhibitory therapeuticsproducing for AD [16,17]. Thebimodal introduction of LA cholinesterase into ChIs has also been used to alter for AD [16,17]. Theimprove introduction LA into ChIs has also been used to alter and Ferulic considerably improve and considerably ChE of inhibition and isoenzyme selectivity [16,17]. acid (FA) and ChE inhibition and isoenzyme selectivity [16,17]. Ferulic acid (FA) and ester derivatives are also its ester derivatives are also attractive candidates for incorporation intoits a hybrid design. These are attractive candidates for incorporation into a hybrid design. Thesedue aretopolyphenolic compounds polyphenolic compounds with potent antioxidant activity in vitro, their ability to scavenge with potent activity in vitro, due to ability tocytoprotective scavenge radicals [18]. They also radicals [18].antioxidant They have also been shown to their up-regulate enzymes such have as heme been shown to up-regulate cytoprotective enzymes such as heme oxygenase-1 and heat shock protein oxygenase-1 and heat shock protein 70, and subsequently inhibit oxidative stress and cell death in 70, and subsequently inhibit oxidative stress cell death with in neurons treated beta amyloid neurons treated with beta amyloid peptide [19].and Pretreatment ferulic acid in awith mouse model was peptide [19].toPretreatment with damage ferulic acid in a mouse model was to inhibit oxidative also shown inhibit oxidative induced memory deficit in also vivo shown [20]. This evidence, taken damage induced memory deficit in vivo [20]. This acid evidence, together, suggested that ester together, suggested that ester derivatives of ferulic couldtaken be brain accessible, multifunctional derivatives of acid could be brain accessible, multifunctional compounds for theincorporation treatment of compounds forferulic the treatment of AD. Several examples have been reported of successful AD. Several of ferulic acidexamples into ChIshave [21].been reported of successful incorporation of ferulic acid into ChIs [21]. The present study concerns the design of neuroprotective hybrids that could selectively inhibit AChE and and modulate modulate ROS ROS damage damage associated. associated. Our design design for for accomplishing accomplishing BuChE BuChE BuChE over AChE specific inhibitors with additional anti-oxidant activity relies on observations around some carbamates have reported reported previously. previously. Isosorbide-2-alkyl Isosorbide-2-alkyl and aryl carbamates carbamates are selective selective and potent potent inhibitors inhibitors we have Figure 1, 1, IC IC50 50 BuChE 4.3 nM), and they are selective for BuChE over AChE and of BuChE (e.g., 1 in Figure related carboxylesterases carboxylesterasesincluding includingCE1 CE1and andCE2 CE2 found human liver intestine Whereas related found in in human liver andand intestine [22].[22]. Whereas the the 2-carbamate is essential to activity, we showed that5-position the 5-position the isosorbide scaffold 2-carbamate is essential to activity, we showed that the of the of isosorbide scaffold could could be modified in diverse producing less potent compounds We hypothesised be modified in diverse ways ways producing more more or lessorpotent compounds [23]. [23]. We hypothesised that that it might be possible to retain the inhibitory isosorbide-2-carbamate functionality but substitute it might be possible to retain the inhibitory isosorbide-2-carbamate functionality but substitute the the effective 5-benzoate 1 with lipoic acid ferulicacid acidesters, esters,retaining retainingcholinesterase cholinesterase sub-type sub-type effective 5-benzoate in 1inwith lipoic acid oror ferulic selectivity while introducing new new disease disease modifying modifying functionality functionality as as shown shown in in Figure Figure1. 1.
Ph
R NH Maintain O 2-carbamate O 2
NH O O
H 2
O
1
O 5
H
O
O Ph
O
S S H
COOH
O 5
H
Replace with antioxidant ester
2
O
O R'
MeO HO
COOH
3
Figure 1. 1. Isosorbide-based carbamates e.g., 1 are potent selective inhibitors of BuChE; formulae for acid (3). (3). lipoic acid (2) and ferulic acid
2. Results and Discussion 2.1. Synthesis of Isosorbide-Based Carbamate-Antioxidants Isosorbide-2-carbamate-5-nitrates (5a–d) were obtained by direct carbamylation of isosorbide mononitrate with the appropriate isocyanate in pyridine. The 2-carbamates 6a–d were obtained by
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2. Results and Discussion 2.1. Synthesis of Isosorbide-Based Carbamate-Antioxidants Isosorbide-2-carbamate-5-nitrates (5a–d) were obtained by direct carbamylation of isosorbide mononitrate with the appropriate isocyanate in pyridine. The 2-carbamates 6a–d were obtained Molecules 2016, 21, 440 3 of 11 by removal of the nitrate group under reductive conditions (H2 , Pd/C). For this work, we chose benzyl carbamate in the isosorbide-2-carbamate-5-ester already reported removal of the because, nitrate group under reductive conditions (H2, Pd/C).compounds For this work, we chose benzyl [22], benzyl carbamates were most potent irrespective of the identity of the 5-ester. Indeed the most potent carbamate because, in the isosorbide-2-carbamate-5-ester compounds already reported [22], benzyl compound we reported was a 2-benzyl carbamate with a 5-salicylate group which had an IC50 for carbamates were most potent irrespective of the identity of the 5-ester. Indeed the most potent compound a 2-benzyl carbamate a 5-salicylate an IC50 for the BuChE of
