J. Med. Chem. 2009, 52, 7249–7257 7249 DOI: 10.1021/jm900628z
Neuroprotective and Cholinergic Properties of Multifunctional Glutamic Acid Derivatives for the Treatment of Alzheimer’s Disease Mariana P. Arce,§ Marı´ a Isabel Rodrı´ guez-Franco,§ Gema C. Gonz alez-Mu~ noz,§ Concepci on Perez,§ Beatriz L opez,§ † † † ,§ Mercedes Villarroya, Manuela G. L opez, Antonio G. Garcı´ a, and Santiago Conde* § Instituto de Quı´mica M edica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain, and †Instituto Te ofilo Hernando, Departamento de Farmacologı´a y Terap eutica, Facultad de Medicina, Universidad Aut onoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain
Received April 28, 2009
Novel multifunctional compounds have been designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimer’s disease (AD). With an L-glutamic moiety as a suitable biocompatible linker, three pharmacophoric groups were joined: (1) an N-benzylpiperidine fragment selected to inhibit acetylcholinesterase by interacting with the catalytic active site (CAS), (2) an N-protecting group of the amino acid, capable of interacting with the acetylcholinesterase (AChE)-peripheral anionic site (PAS) and protecting neurons against oxidative stress, and (3) a lipophilic alkyl ester that would facilitate penetration into the central nervous system by crossing the blood-brain barrier. At submicromolar concentration, they inhibit AChE and butyrylcholinesterase (BuChE) of human origin, displace the binding of propidium iodide from the PAS of AChE, and could thus inhibit Aβ aggregation promoted by AChE. They also display neuroprotective properties against mitochondrial free radicals, show low toxicity, and could be able to penetrate into the CNS.
*To whom correspondence should be addressed. Phone: þ34 91 5622900. Fax: þ34 91 5644853. E-mail:
[email protected].
reactive oxygen species (ROS). In addition, the brain requires metals to carry out many of its functions, and most ROS are formed as byproducts in redox reactions of molecular oxygen with some active metals.6 Aβ displays high affinity for redox metal ions and exhibits antioxidant properties by reducing them, contributing to regulation of the redox balance.7 However, abnormal metabolism of Aβ turns it into a neurotoxic species when small aggregates are formed. Soluble oligomers or protofibrils are present in the formation of hydrogen peroxide and other oxidative species, which increase the oxidation of lipids, DNA, and proteins, producing mitochondrial dysfunction.8,9 Therefore, antioxidants and inhibitors of Aβ aggregation appear as interesting candidates to treat AD in its earlier phases. Another physiological function affected in AD is cholinergic neurotransmission, which seems to be closely related to pathological Aβ.10 Currently, the cholinergic strategy remains the most effective therapeutic approach for the symptomatic treatment of AD.11,12 This hypothesis asserts that most of the cognitive impairments suffered by AD patients are the consequence of a deficit in acetylcholine (ACh) and thus in cholinergic neurotransmission. Therefore, inhibition of acetylcholinesterase (AChE) appears to be a useful therapeutic path to reduce, at least temporarily, the cognitive deficit in AD. To date, most of the drugs available in the market for the treatment of AD are acetylcholinesterase inhibitors (AChEI).13 In addition, increasing attention is being dedicated to butyrylcholinesterase (BuChE), an enzyme also involved in the cholinergic neurotransmission.14 In healthy brains, AChE hydrolyzes about 80% of acetylcholine, while BuChE plays a secondary role. In AD brains, the activity of AChE decreases, while that of BuChE rises in an attempt by the neurons to modulate the ACh levels. So, both enzymes are
r 2009 American Chemical Society
Published on Web 10/26/2009
Introduction As a consequence of the progressive aging of the world population, diseases associated with age, such as senile dementias, are gradually increasing their importance. Alzheimer’s disease (AD) is the most widespread dementia, accounting for 60-80% of all cases, followed by vascular dementias.1 Currently there are about 17 million AD patients worldwide, and it is predicted that this figure will reach 70 million by 2050 if an efficient treatment is not developed.2 The effects of AD are devastating both for patients and for caregivers, and the costs for the corresponding Public Health System and for families are very high. The well-known distinctive characteristics of AD, that is, deposits of amyloid β-peptide (Aβ), neurofibrillary tangles, massive loss of neurons, and severe cognitive impairment are late-stage features of the disease, probably the final phase of a complex pathological process. Long before, several connected biochemical reactions have been undergoing abnormal changes in their physiological functions, which finally develop the symptoms, histopathological hallmarks, and death. Many aspects of AD remain unclear, in particular the initial cause or causes and the relationships between the damaged processes. Despite this, there is increasing evidence that oxidative imbalance and the consequent oxidative stress and changes in the functional production of Aβ are at the earliest steps of the disease.3,4 Although the brain constitutes only a mere 2% of the total body weight, it is a major oxygen-consuming organ, using around 20% of the resting body’s oxygen.5 Therefore, the oxidative balance must be tightly controlled by antioxidants in order to eliminate the oxidative species such as
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7250 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Arce et al.
Scheme 1. Synthesis of 1a, 1c, and 1d-ia
a
Figure 1. Three groups linked by the L-glutamic acid chain in a single molecule.
implicated in the regulation of ACh and are therapeutic targets when confronting the cholinergic deficit. Inhibitors of both, such as rivastigmine, have proven clinical efficacy.15 In addition, there is also increasing evidence that AChE exhibits noncholinergic functions related to neuronal development, differentiation, and adhesion. AChE also plays an important role in the processing of Aβ through the peripheral anionic site (PAS) of the enzyme that interacts with Aβ oligomers and fibrils, promoting their aggregation.16,17 This effect is produced by electronic interactions of the Trp279 residue located in the PAS with an electron-rich moiety of the molecule, such as aromatic or carbamate groups.18 These facts have led to the study of compounds showing a dual activity as inhibitors of both AChE and Aβ aggregation, because they can simultaneously improve cognition and slow the rate of Aβ degeneration.19 Currently, the interest in these dual-site inhibitors has increased. As an example, NP-61 is the first compound of this class in phase I clinical trials for AD.20 In recent years, the multifaceted condition of AD has promoted an active search for multifunctional drugs with two or more selected biological activities, since they may represent an important pharmacological advance in the management of the disease.21,22 Continuing with our research on various heterocyclic compound families with potential application in AD,23-27 we recently reported the synthesis of multifunctional drugs that combine dual inhibition of AChE and neuroprotective properties in a single small molecule.28,29 At present, our work is focused on the design of new multiactivity compounds in which different active units could be anchored to a biocompatible scaffold. Dicarboxylic amino acids could be considered as interesting biological carriers of pharmacophoric groups because they present three points (two carboxylic acids and one amine) to anchor them. Several years ago, we published an exploratory study of some R-amides of L-glutamic acid and ω-amides of S-R-aminoadipic and R,S-R-aminopimelic acids obtained by enzymatic catalysis in organic solvents.30 In that paper, we described the good AChEI activity displayed by the ω-amides, which contrasted with the lack of activity of R-amides. These results prompted us to obtain new derivatives and carry out a broader evaluation. In this paper, we present the first study on the synthesis and biological activity of a new series of multifunctional compounds in which three active groups are joined to L-glutamic acid as a suitable biological linker. The three pharmacophoric
Reagents and conditions: (i) nHexOH, TEA, CH2Cl2, PyBOP; (ii) H2, Pd/C, MeOH; (iii) R0-NH2, EDC, HOBt, DMAP, TEA, dry DMF; (iv) TFA, CH2Cl2; (v) R-CO2H, EDC, HOBt, DMAP, TEA, dry DMF; (vi) R-COCl, TEA, CH2Cl2.
groups are (a) an ω-situated N-benzylpiperidine moiety able to bind the catalytic active site (CAS) of AChE, (b) an N-protecting group, capable of interacting with the PAS in order to inhibit Aβ aggregation and, tentatively, able to display neuroprotective activity, and finally, (c) a lipophilic R-hexyl ester that could favor crossing of the blood-brain barrier (BBB)31 (Figure 1). L-Glutamic acid was chosen because it has the appropriate distance between RNH and γCO2H to allow simultaneous interaction between the pharmacophoric fragments and the two main AChE-sites, namely, the CAS and PAS. L-Glutamic acid also presents the additional advantage of the existence of a high number of commercially available derivatives to be taken as synthetic starting materials. The pharmacological evaluation of these novel compounds includes the inhibition of AChE and BuChE, displacement of propidium iodide from AChE, as a preliminary method to test their potential inhibition of Aβ aggregation, in vitro brain penetration, and cell viability. We also studied the neuroprotective effects of these derivatives against death induced in human neuroblastoma cells by various toxic insults related to oxidative stress triggered by hydrogen peroxide and the mixture of rotenone and oligomycin A. Chemistry. The syntheses of the desired compounds started from enantiomerically pure commercial derivatives, following well-known methods in peptide chemistry (Scheme 1). N-Boc-L-Glu(OBn)-OH was esterified with 1-hexanol (nHexOH) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). The resulting diester 2 was hydrogenated obtaining the free ω-acid 3. Then, it was coupled with commercial N-benzyl-4-(2-aminoethyl)piperidine (R0-NH2) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxybenzotriazole (HOBt) and 4-(dimethylamino)pyridine (DMAP) to yield 1a, which was taken both as a final product and as an intermediate compound when it was unblocked with trifluoroacetic acid (TFA) to yield the ammonium salt 4. This compound was first amidated with 2-(6-chlorobenzo[b]thiophen-2-yl)acetic acid using EDC, HOBt, and DMAP as coupling reactants, affording 1c but in poor yield. In order to improve this point, classical amidation with acyl chlorides was used, obtaining 1d-i in satisfactory yields. The compound 1b was synthesized from N-Cbz-L-Glu(OtBu)-OH and esterified with nHexOH yielding the diester nHex-tBu 5. Then, the tBu moiety was eliminated with TFA resulting in the free ω-acid 6 that finally was amidated with R0-NH2 to yield 1b (Scheme 2).
Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
7251
Scheme 2. Synthesis of 1ba
a
Reagents and conditions: (i) nHexOH, TEA, PyBOP, dry CH2Cl2; (ii) TFA, CH2Cl2; (iii) R0-NH2, EDC, HOBt, DMAP, TEA, dry DMF.
Table 1. Inhibition of AChE and BuChE (IC50, μM)a by the new compounds, 1a-i compd
AChEb
BuChEc
h-AChEd
h-BuChEd
selectivity h-AChE vs h-BuChE
1a 1b 1c 1d 1e 1f 1g 1h 1i tacrine
5.00 ( 0.10 1.40 ( 0.40 2.30 ( 0.10 1.90 ( 0.10 3.50 ( 0.10 1.20 ( 0.10 2.50 ( 0.20 0.33 ( 0.01 3.50 ( 0.20 0.040 ( 0.002
3.50 ( 0.30 0.75 ( 0.03 0.05 ( 0.01 1.50 ( 0.10 1.00 ( 0.10 0.75 ( 0.50 0.55 ( 0.01 0.95 ( 0.03 0.70 ( 0.04 0.010 ( 0.001
0.95 ( 0.10 0.25 ( 0.01 0.10 ( 0.01 0.53 ( 0.02 0.50 ( 0.02 0.18 ( 0.01 0.27 ( 0.02 0.44 ( 0.08 0.30 ( 0.02 0.35 ( 0.01
3.00 ( 0.10 0.73 ( 0.02 0.07 ( 0.01 1.75 ( 0.60 1.80 ( 0.10 0.85 ( 0.04 0.33 ( 0.01 0.83 ( 0.07 0.35 ( 0.01 0.040 ( 0.002
3.2 2.9 0.7 3.3 3.6 4.7 1.2 1.9 1.2 0.1
a Mean of three independent measurements ( SEM (standard error of the mean). b Bovine erythrocyte AChE. c Horse serum BuChE. d Human enzymes.
Cholinesterase Inhibition. The new L-glutamic acid derivatives 1a-i were evaluated as AChE and BuChE inhibitors, following the method described by Ellman,32 using tacrine as reference. Because of their lower cost, enzymes of animal origin were initially used: AChE from bovine erythrocytes and BuChE from horse serum. All the compounds showed good inhibition of these enzymes, displaying IC50’s in the micromolar range (Table 1). Then, they were checked on enzymes of human origin, and the results are also gathered in Table 1. All derivatives inhibited the human AChE (h-AChE) 10-fold more efficiently than the bovine enzyme, showing IC50 values in the submicromolar range (0.100.53 μM). They also inhibited human BuChE (h-BuChE) with IC50’s around the micromolar range, exhibiting thus a moderate selectivity toward h-AChE. Propidium Displacement Assay as a Probe of the Inhibition of Aβ Aggregation. As mentioned in the Introduction, AChE interacts with Aβ oligomers and fibrils and contributes to their aggregation. The structural motif of the enzyme that promotes Aβ fibril formation is located at the PAS, where Trp279 appears to play an important role.33 This hypothesis is supported by studies demonstrating that selective ligands that bind to the PAS, such as propidium iodide, are able to block Aβ aggregation.34 Thus, the ability of this new series to bind to the PAS of AChE is an interesting point to investigate because, in case of positive results, the compounds could be potential good candidates for the development of new antiAβ drugs. The affinity of new compounds 1a-i for the PAS was studied by displacement of propidium iodide, a specific AChE PAS ligand that exhibits a 10-fold higher fluorescence when bound to AChE.35 A decrease in propidium fluorescence in the presence of a tested compound could be interpreted as a measure of the affinity of such compound for the PAS, displacing the propidium cation from it. Compounds 1a-i were evaluated at three concentrations, 0.3, 1, and 3 μM, and the results are gathered in Table 2. The majority of the compounds showed significant abilities to displace propidium cation from the PAS of AChE, with values higher than 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), a selective ligand
Table 2. Displacement of Propidium Iodide from the Peripheral Anionic Site of AChE by the New Compounds 1a-i and BW284c51 as Reference, at the Indicated Concentrationsa compd
0.3 μM
1 μM
3 μM
1a 1b 1c 1d 1e 1f 1g 1h 1i BW284c51
42.3 ( 6.5 20.8 ( 2.5 14.7 ( 4.0 24.7 ( 7.2 7.5 ( 1.0 11.3 ( 2.2 31.5 ( 3.1 37.4 ( 3.8 42.2 ( 5.7 b
43.6 ( 8.6 25.0 ( 3.3 16.0 ( 2.8 15.9 ( 3.2 10.3 ( 1.4
