Neuroprotective and cholinergic properties of multifunctional glutamic acid derivatives for the treatment of Alzheimer's disease

Article abstract of DOI:10.1021/jm900628z

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) anN

Full text of DOI:10.1021/jm900628z

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 Rodrıguez-Franco, Gema C. Gonzalez-Munoz, Concepcion Perez, Beatriz Lopez,
ꢀ ~ ꢀ ꢀ ꢀ
´ ´
´
a,† and Santiago Conde*,§
†
†
ꢀ
Mercedes Villarroya, Manuela G. Lopez, Antonio G. Garcı
§
†
Instituto de Quımica Medica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain, and Instituto Teofilo Hernando, Departamento de
´
Farmacologıa y Terapeutica, Facultad de Medicina, Universidad Autonoma 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.
Introduction
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 How-
ever, 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 mitochon-
drial 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 choliner-
gic 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 con-
sequence of a deficit in acetylcholine (ACh) and thus in
cholinergic neurotransmission. Therefore, inhibition of acetyl-
cholinesterase (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 butyryl-
cholinesterase (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
As a consequence of the progressive aging of the world
population, diseases associated with age, such as senile de-
mentias, are gradually increasing their importance. Alzhei-
mer’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 im-
balanceandthe consequentoxidative stress and changesin 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 anti-
oxidants in order to eliminate the oxidative species such as
*To whom correspondence should be addressed. Phone: þ34 91
5622900. Fax: þ34 91 5644853. E-mail: sconde@iqm.csic.es.
r
2009 American Chemical Society
Published on Web 10/26/2009
pubs.acs.org/jmc

7250 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Arce et al.
Scheme 1. Synthesis of 1a, 1c, and 1d-ia
a 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.
Figure 1. Three groups linked by the L-glutamic acid chain in a
single molecule.
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 phar-
macophoric fragments and the two main AChE-sites, namely,
the CAS and PAS. L-Glutamic acid also presents the addi-
tional advantage of the existence of a high number of com-
mercially available derivatives to betaken 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 prelimin-
ary method to test their potential inhibition of Aβ aggrega-
tion, 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 per-
oxide and the mixture of rotenone and oligomycin A.
Chemistry. The syntheses of the desired compounds star-
ted 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 (nHex-
OH) 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)piperi-
dine (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 com-
pound 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 synthe-
sized 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).
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 deve-
lopment, 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 carbamategroups.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 in-
hibitors 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 man-
agement of the disease.21,22 Continuing with our research on
various heterocyclic compound families with potential appli-
cation 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 multi-
activity 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-aminoadi-
pic 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
promptedustoobtainnew derivatives andcarry outabroader
evaluation.
In this paper, we present the first study on the synthesis and
biological activity of a new series of multifunctional com-
pounds in which three active groups are joined to L-glutamic
acid as a suitable biological linker. The three pharmacophoric

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
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
1c
1d
1e
1f
1g
1h
1i
tacrine
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 deriva-
tives 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 (0.10-
0.53 μM). They also inhibited human BuChE (h-BuChE)
with IC50’s around the micromolar range, exhibiting thus a
moderate selectivity toward h-AChE.
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
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
<0.5
42.0 ( 8.2
29.3 ( 3.2
23.4 ( 4.6
37.3 ( 5.2
15.0 ( 2.0
<0.5
1c
1d
1e
1f
1g
26.5 ( 6.1
35.4 ( 5.9
33.3 ( 9.1
b
39.7 ( 5.7
27.2 ( 8.1
23.3 ( 4.4
16.0 ( 2.4
1h
1i
BW284c51
a Results are the mean ( SEM (n = 8). b Not determined.
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 anti-
Aβ 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 fluores-
cence in the presence of a tested compound could be inter-
preted 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.
used as reference in these assays.36 In some cases, nonlinear
concentration-response data were obtained, which could be
explained by some solubility problems or by the fact that
these multifunctional compounds could interact with other
points of the gorge. These results suggest that the new
glutamic acid derivatives 1a-i are able to bind to AChE-
PAS, and could therefore inhibit Aβ fibril formation pro-
moted by this enzyme.16
In Vitro Blood-Brain Barrier Permeation Assay. Screen-
ing for blood-brain barrier (BBB) penetration in the first
steps of drug discovery research is very important, since the
first requirement for successful CNS drugs is to penetrate the
BBB and reach their therapeutic targets. In this preliminary
work, we used a parallel artificial membrane permeation
assay for blood-brain barrier (PAMPA-BBB) to explore
whether these glutamic acid derivatives would be able to
penetrate into the brain. This simple and rapid model,
described by Di et al.37 and successfully applied by us to
different compounds,28,29,38-41 has the advantage of predict-
ing passive BBB permeation with high success. The in vitro
permeabilities (Pe) of the glutamic acid derivatives herein
described and 15 commercial drugs through a lipid extract of
porcine brain were determined, and the results are gathered
in Table 3. Assay validation was made comparing the
experimental permeability with the reported values of these
The majority of the compounds showed significant abi-
lities to displace propidium cation from the PAS of AChE,
with values higher than 1,5-bis-(4-allyldimethylammonium-
phenyl)pentan-3-one dibromide (BW284c51), a selective ligand

7252 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Arce et al.
Table 3. Permeability (Pe, 10-6 cm s-1) in the PAMPA-BBB Assay for
15 Commercial Drugs (Used in the Experiment Validation) and the
Glutamic Acid Derivatives 1a-i with their Predicted Penetration in the
CNS
Table 4. Neuroprotection (%) in the Human Neuroblastoma Cell Line
SH-SY5Y against H2O2 (60 μM) at the Indicated Concentrationsa
compd
1 μM
3 μM
10 μM
30 μM
1a
1b
24.9b
4.1
1.7
17.4b
32.5c
68.1d
64.5d
84.3d
20.9
30.7
41.6b
0
0
0
5.3
Pe (10-6
bibla cm s-1 b
Pe (10-6
compd cm s-1 b
39.1c
0
compd
)
)
prediction
1c
1d
26.2
43.9c
55.1d
63.3d
28.7
0
0
52.4d
73.0d
66.7d
21.2
27.9
46.4b
e
67.0d
31.8d
66.7c
17.9
48.8
20.3b
57.7d
testosterone
verapamil
17.0 24.5 ( 2.1
16.0 20.2 ( 1.9
13.0 14.4 ( 0.9
12.0 16.2 ( 0.8
9.3 17.7 ( 0.9
8.8 15.5 ( 1.0
6.5 11.0 ( 0.8
1a
1d
1e
1g
1h
1i
11.6 ( 0.8
CNS þ
CNS þ
CNS þ
CNS þ
CNS þ
CNS þ
18.7 ( 1.6
20.9 ( 1.8
25.6 ( 1.3
18.8 ( 0.9
28.5 ( 2.0
1e
imipramine
desipramine
progesterone
promazine
chlorpromazine
clonidine
1f
1g
1h
1i
trolox
54.5b
0
5.3
5.1
2.5
1.9
1.3
1.1
0.9
0.8
9.5 ( 0.5
6.4 ( 0.1
3.7 ( 0.1
5.0 ( 0.2
4.3 ( 0.1
2.5 ( 0.1
2.6 ( 0.1
2.3 ( 0.1
a Results are the mean ( SEM (n = 4). b p < 0.05. c p < 0.01.
corticosterone
piroxicam
d p<0.001. e Not determined.
hydrocortisone
caffeine
Table 5. Neuroprotection (%) of 1d-f, 1i, and Trolox in the Human
Neuroblastoma Cell Line SH-SY5Y against the Combination of Rote-
none (30 μM) and Oligomycin A (10 μM) at the Indicated Concentra-
tions, Using Pre- and Co-incubation Conditionsa
lomefloxacin
enoxacin
ofloxacin
a Taken from ref 23. b Data are the mean ( SD of three independent
experiments.
preincubation
co-incubation
compd 0.1 μM 0.3 μM 1 μM 3 μM 0.1 μM 0.3 μM 1 μM 3 μM
1d
1e
e
47.9d 43.8d 38.5c
e
e
20.1 21.0 15.1
48.9b
27.1
19.0
22.3
27.8b 21.4
e
0
0
19.7
7
0
e
commercial drugs, which gave a good linear correlation,
Pe(exptl)=1.24Pe(bibl) þ 1.98 (R2=0.93). From this equa-
tion, and taking into account the limits established by Di
et al. for BBB permeation,37 we found that molecules with a
permeability >7.0ꢀ 10-6 cm s-1 would be able to cross the
BBB by passive permeation.
1f
e
19.6
e
63.7d
1i
trolox
34.8c
46.1c 45.4c
e
22.3
e
20.8 30.8b
e
e
e
65.1d
e
e
a Results are the mean ( SEM (n = 4). b p < 0.05. c p < 0.01.
d p<0.001. e Not determined.
All the glutamic acid derivatives showed permeability
values over the above limit, as the known CNS drugs used
in the assay validation, pointing out that molecules herein
described would cross the BBB by passive diffusion
(Table 3).
Cell Viability and Neuroprotection Studies. To gain insight
the into the therapeutic potential of these glutamic acid
derivatives, cell viability and neuroprotective capacity
against oxidative stress were assayed using the human neu-
roblastoma cell line SH-SY5Y. Two different models of
oxidative stress were employed: (i) incubation with hydrogen
peroxide for the generation of exogenous free radicals and
(ii) incubation with a combination of rotenone and oligo-
mycin A for the induction of mitochondrial ROS, as a
consequence of the blockade of complexes I and V of the
electron transport chain.42
In the first model, cells were incubated with compounds
1a-i at four concentrations (1, 3, 10, and 30 μM) during 24 h
before the addition of 60 μM hydrogen peroxide, and then
they were maintained for another 24 h period. After that, the
presence of lactic dehydrogenase (LDH) was measured as a
parameter of cell death, since this enzyme is released to the
extracellular medium when neurons die.43 Basal release of
LDH by the cells was subtracted from the values obtained for
all compounds in order to calculate the percentage of
neuroprotection. Trolox, the vitamin E antioxidant moiety,
was used as a positive control, and results are shown in
Table 4.
All tested compounds 1a-i protected cells from damage
induced by H2O2, sometimes better than trolox. Four deri-
vatives (1d-1f, 1i) displayed significant and interesting
percentages of neuroprotection, above 50% in almost
all tested concentrations. In particular, compounds 1f and
1e showed the higher protection values, 84% at 3 μM and
73% at 10 μM, respectively. It is worth-mentioning that
neuroprotection is very frequently lost at higher product
concentrations, probably due to solubility problems or to
additional interactions with other biological targets. In fact,
some of us have found this behavior in some marketed
compounds like galantamine or donepezil, which show a
characteristic U shape concentration-dependent curve for
their neuroprotective action.44,45
Possible cytotoxic effects of 1a-i were studied by exposing
cells to compounds at the highest concentration used in the
neuroprotection studies (30 μM) for 24 h. In all cases, LDH
percentage was lower than or equal to the basal value,
pointing out that cell viability reached 100%.
The neuroprotective effect of compounds 1d, 1e, 1f, and 1i
against mitochondrial oxidative stress was also evaluated by
using a combination of rotenone (30 μM) and oligomycin A
(10 μM) as the toxic insult in neuroblastoma cells. Two
different protocols were used: (i) Preincubation, wherein
cells were incubated with the four compounds during 24 h
before the addition of rotenone and oligomycin A, and then
maintained for an additional 24 h period. This protocol has
been used in our group in the last years to assess a possible
cytoprotective effect due to activation of endogenous anti-
oxidant pathways.46-48 (ii) Co-incubation, wherein the com-
pounds and the combination rotenone plus oligomycin A
were added at the same time and incubated for 24 h. In this
case, a cytoprotective effect would indicate that the com-
pounds could be acting as free radical scavengers. Results are
shown in Table 5.
When preincubated, the compounds display good levels of
protection, with the highest values of nearly 50% exhibited
by 1d, 1e, and 1i at the lowest concentrations of 0.3, 0.1, and
0.3 μM respectively. As a counterpart, protection was in
general lower when the cells were simultaneously co-incu-
bated with the compounds and the combination of rotenone

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7253
plus oligomycin A. These different results suggest that the
protective mechanism of these new L-glutamic acid deriva-
tives might be mainly due to the expression of antioxidant
proteins or the activation of intracellular signaling pathways
involved in cell survival, elicited by the long-term preincuba-
tion of the cells with the compounds. Further experiments
are needed to search the exact neuroprotective mechanism.
Thus, the compounds of this new family of glutamic acid
derivatives have proven to be neuroprotectants against both
exogenous and mitochondrial ROS. Such properties could
decrease oxidative stress, which is a characteristic of neuro-
degenerative illnesses like Alzheimer’s disease.
sulfate and evaporated to dryness under reduced pressure. The
residue was purified by flash column chromatography eluting
with Hex/AcOEt (6:1) obtaining an oil. Yield: 1.2 g (93%). MS
(ESIþ) m/z=444 [M þ H]þ. 1H NMR (400 MHz, CDCl3) δ 7.35
(m, 5H), 5.11 (s, 2H), 5.09 (s, 1H), 4.33 (m, 1H), 4.11 (m, 2H),
2.43 (m, 2H), 2.20 (m, 1H), 1.96 (m, 1H), 1.62 (m, 2H), 1.43
(s, 9H), 1.27-1.33 (m, 6H), 0.88 (t, J=7.0 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 172.5, 172.2, 155.3, 135.0, 128.5 (2C),
128.3, 128.2 (2C), 80.0, 66.6, 65.7, 52.9, 31.3, 30.3, 28.4, 28.3
(3C), 27.9, 25.5, 22.5, 14.0. Purity: 99% (by HPLC). Anal.
(C23H35NO6) C, H, N.
N-Boc-L-Glu(OH)-O-nHex (3): A solution of 2 (1.1 g, 3.3 mmol)
in MeOH (30 mL) was hydrogenated at 18 psi in the presence of
10% palladium/charcoal (100 mg) at room temperature. After 3 h,
the catalyst was filtered off, and the solvent was evaporated at
reduced pressure. The residue was purified by flash column
chromatography eluting with Hex/AcOEt (2:1), affording a white
solid. Yield: 840 mg (98%). Mp: 42-45 °C. MS (ESIþ) m/z=332
[M þ H]þ. 1H NMR (400 MHz, CDCl3) δ 10.77 (s, 1H), 5.64 (m,
1H), 4.41 (m, 1H), 4.12 (m, 2H), 2.40 (m, 2H), 2.16 (m, 1H), 1.94
(m, 1H), 1.60 (m, 2H), 1.44 (s, 9H), 1.29-1.21 (m, 6H), 0.88 (t, J=
6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.6, 172.2, 156.1,
80.0, 65.7, 53.2, 31.2, 29.9, 28.4 (3C), 28.1, 27.3, 25.3, 23.8, 13.8.
Purity: g 99% (by HPLC). Anal. (C16H29NO6) C, H, N.
N-Boc-L-Glu[NH-2-(1-benzylpiperidin-4-il)ethyl]-O-nHex (1a):
N-Benzyl-4-(2-aminoethyl)piperidine (143 mg, 0.65 mmol),
EDC (139 mg, 0.72 mmol), HOBt (111 mg, 0.72 mmol), TEA
(275 μL, 1.95 mmol), and DMAP (8 mg, 0.065 mmol) were
added to a solution of 3 (240 mg, 0.72 mmol) in dry dimethyl-
formamide (DMF) (6 mL) and stirred for 18 h at room tem-
perature. Then, the solvent was evaporated, and the residue was
purified by silica gel column chromatography, eluting with
CH2Cl2/MeOH (95:5), affording 1a as a colorless oil (265 mg,
69% yield). [R]D20: -22.84 (c 1.2, MeOH). MS (ESIþ) m/z=532
[M þ H]þ.1H NMR (400 MHz, CDCl3) δ 7.40 (m, 5H), 6.46 (bs,
1H), 5.37 (m, 1H), 4.18-4.07 (m, 3H), 3.37 (s, 2H), 3.22 (m, 2H),
2.85 (m, 2H), 2.40-2.22 (m, 4H), 1.90 (m, 2H), 1.61-1.52 (m,
4H), 1.40 (s, 9H), 1.34-1.27 (m, 11H), 0.87 (t, J = 6.8 Hz,
3H).13C NMR (100 MHz, CDCl3) δ 172.6, 172.3, 155.9, 131.8,
130.8 (2C), 129.8 (2C), 129.2, 80.0, 65.7, 61.3, 53.1 (2C), 52.6,
37.5, 36.6, 34.7, 32.2, 31.3 (2C), 30.7, 28.6, 28.4, 28.2 (3C),
25.4, 22.4, 14.0. Purity: 98% (by HPLC). Anal. (C30H49N3O5)
C, H, N.
L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl)-O-nHex, trifluoro-
acetic salt (4): A solution of 1a (265 mg, 0.49 mmol) and TFA
(547 μL) in CH2Cl2 (2 mL) was stirred for 6 h. Then, the solvent
was evaporated, obtaining 4 as yellow oil (260 mg, 96%). MS
(ESIþ) m/z=432 [M þ H]þ.1H NMR (300 MHz, MeOD) δ 7.38
(s, 5H), 4.11 (m, 2H), 4.13 (s, 2H), 3.25 (m, 1H), 3.17 (m, 2H),
2.93 (m, 2H), 2.28 (m, 2H), 2.15 (m, 2H), 2.01 (m, 1H), 1.85 (m,
1H), 1.78 (m, 2H), 1.64 (m, 2H), 1.42-1.24 (m, 11H), 0.89 (t, J=
6.8 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 174.8, 173.7, 135.9,
130.2 (2C), 130.0 (2C), 128.3, 65.2, 62.8, 53.6, 53.3 (2C), 36.8,
35.8, 33.0, 31.9, 31.5 (2C), 31.2, 30.3, 28.6, 25.6, 22.5, 13.3.
Purity: 98% (by HPLC). Anal. (C27H42F3N3O5) C, H, N.
N-6-Chlorobenzo[b]thieno-2-yl-acetyl-L-Glu[NH-2-(1-benzyl-
piperidin-4-yl)ethyl]-O-nHex (1c): 2-(6-Chlorobenzo[b]thiophen-
2-yl)acetic acid (62 mg, 0.28 mmol), EDC (52 mg, 0.28 mmol),
HOBt (42 mg, 0.28 mmol), TEA (275 μL, 0.75 mmol), and DMAP
(8 mg, 0.02 mmol) were addedtoasolutionof4(137 mg, 0.25 mmol)
in dry DMF (6 mL) and stirred for 18 h at room temperature. Then,
the solvent was evaporated, and the residue was purified by silica gel
Conclusions
This first work presents a new series of L-glutamic acid
derivatives that exhibit attractive in vitro biological activities.
At submicromolar concentrations, they efficiently inhibit
acetylcholinesterase (AChE), displace propidium from the
PAS of the AChE, and protect neurons against damage
caused by both exogenous and mitochondrial free radicals.
Finally, they have no toxicity and would be able to penetrate
in the CNS by passive diffusion to reach their cerebral targets.
It is consequently expected that these multifunctional com-
pounds could be able to improve cognitive impairment,
diminish the oxidative damage caused by free radicals, includ-
ing those of mitochondrial origin, and delay the degenerative
process related to the excessive deposition of Aβ in patients
suffering from Alzheimer’s disease. Such interesting proper-
ties highlight these new compounds as good candidates for
further studies directed to the development of new multi-
functional drugs useful in the treatment of Alzheimer’s dis-
ease.
Experimental Section
Chemistry. General Methods. Reagents were purchased from
common commercial suppliers and were used without further
purification. Solvents were purified and dried by standard
procedures. Chromatographic separations were performed on
silica gel using flash-column chromatography (Kieselgel 60,
Merck, 230-400 mesh), and compounds were detected with
UV light (λ=254 nm). Nuclear magnetic resonance spectra were
recorded using a Varian XL-300 and Varian Unity Inova-400
spectrometers. Chemical shifts are reported in δ values (ppm)
relative to internal Me4Si, J values are reported in hertz, and
spin multiplicities are expressed as s (singlet), bs (broad singlet),
d (doublet), t (triplet), or m (multiplet). HPLC analyses were
performed on Alliance Waters 2690 equipment, with a UV
detector photodiode array Waters 2996 with MS detector
MicromassZQ (Waters), using an XBridge C18, 3.5 μm, 200 A
(2.1 nmꢀ100 mm) column. The standard gradient consisted of a
10 min run from 5% to 100% of acetonitrile at a flow rate of
0.25 mL/min. Mass spectra (MS) were obtained by electron
spray ionization (ESI) in positive mode using a Hewlett-Packard
MSD 1100 spectrometer. Elemental analyses were carried out in
Perkin-Elmer 240C equipment. Optical rotations were deter-
mined with a Perkin-Elmer 241 MC polarimeter at room
temperature (ca. 295 K). Melting points were determined in a
Reichert-Jung Thermovar apparatus.
column chromatography, eluting with CH2Cl2/MeOH (95:5), to
N-Boc-L-Glu(OBn)-O-nHex (2): PyBOP (1.7 g, 3.29 mmol),
n-hexanol (367 mg, 3.59 mmol), and triethylamine (TEA)
(830 μL, 5.99 mmol) were added to a solution of N-Boc-L-
Glu(OBn)-OH (1.0 g, 2.99 mmol) in dry CH2Cl2 (60 mL) and
stirred for 16 h at 0 °C. Then, more CH2Cl2 (50 mL) was added,
and the final solution was washed with diluted HCl (3ꢀ30 mL),
then with a 10% aq solution of NaOH (3ꢀ30 mL), and finally
with brine (3ꢀ30 mL). The organic phase was dried over sodium
20
afford 1c as a yellow solid (46 mg, 28%). Mp: 84-86 °C. [R]D
:
-25.43 (c 1, MeOH). MS (ESIþ) m/z=641 [M þ H]þ. 1H NMR
(300 MHz, MeOD) δ 7.80 (m, 3H), 7.55 (s, 1H), 7.34 (m, 5H), 4.39
(m, 1H), 4.08 (m, 2H), 3.81 (s, 2H), 3.72 (s, 2H), 3.18 (m, 2H), 3.00
(m,2H), 2.31(m, 3H), 2.17(m, 2H),1.93(m, 1H),1.73(m, 2H),1.54
(m, 2H), 1.39 (m, 2H), 1.33-1.25 (m, 9H), 0.84 (t, J=7.0 Hz, 3H).
13C NMR (75 MHz, MeOD) δ 174.3, 173.2, 172.7, 141.5, 140.0,

7254 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Arce et al.
135.2, 131.6, 131.4, 130.0 (2C), 129.6 (2C), 129.5, 128.0, 125.8, 125.1,
122.8, 66.4, 63.1, 54.0, 53.0 (2C), 39.7, 37.6, 36.3, 33.0, 32.5, 31.6
(2C), 29.6, 28.3, 26.7, 26.5, 23.6, 14.4. Purity: 99% (by HPLC). Anal.
(C35H46ClN3O4S) C, H, N, S.
(2C), 128.5 (2C), 127.5, 126.6, 125.7, 125.4, 125.1, 123.0, 66.1,
63.4, 53.7, 53.2 (2C), 37.6, 36.2, 33.3, 33.0, 31.9 (2C), 31.6, 28.7,
27.7, 25.7, 22.7, 14.2. Purity: 98% (by HPLC). Anal.
(C34H45N3O4S) C, H, N, S.
General Procedures for Synthesis of Acyl Chlorides. The
selected acid was refluxed in thionyl chloride for 4-6 h, and
then the solution was cooled to room temperature, and the
remaining thionyl chloride was evaporated to afford the acyl
chloride, which was used without further purification.
General Procedures for the Synthesis of L-Glutamic Deriva-
tives 1d-i. A solution of the acyl chloride in CH2Cl2 (2 mL)
was added dropwise to a solution of 4 (0.20 mmol) and TEA
(0.44 mmol) in dry CH2Cl2 cooled to 0 °C. The final solution was
stirred at room temperature for 14 h. Then, the solvent was
evaporated, and the residue was purified by silica gel column
chromatography using a mixture of CH2Cl2/MeOH (95:5).
N-Benzoyl-L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl]-O-nHex (1d):
Reaction of 4 (88 mg, 0.16 mmol), benzoyl chloride (21 μL, 0.17
mmol), and Et3N (54 μL, 0.39 mmol) afforded a colorless solid
(80 mg, 92%). Mp: 89-91 °C. [R]D20: -30.25 (c 0.9, MeOH). MS
(ESIþ) m/z=536 [M þ H]þ. 1H NMR (400 MHz, CDCl3) δ 7.85
(m, 3H), 7.46 (m, 3H), 7.30 (m, 5H), 6.09 (m, 1H), 4.69 (m, 1H),
4.16 (td, J=6.33, 1.93 Hz, 2H), 3.54 (s, 2H), 3.26-3.19 (m, 2H),
2.90 (m, 2H), 2.38-2.17 (m, 4H), 2.12-1.98 (m, 2H), 1.66-1.60
(m, 4H), 1.40-1.25 (m, 11H), 0.87 (t, J=7.0 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 172.2, 172.0, 167.4, 133.5, 131.9, 129.5,
128.6 (2C), 128.3 (3C), 127.3 (2C), 127.1 (2C), 65.9, 63.0, 53.4
(2C), 52.7, 37.3, 35.9, 33.1, 32.8, 31.6 (2C), 31.3, 28.5, 28.1,
25.4, 22.5, 14.0. Purity: 99% (by HPLC). Anal. (C32H45N3O4)
C, H, N.
N-Pyridin[2,3-b]thieno-2-yl-L-Glu[NH-2-(1-benzylpiperidin-4-yl)-
ethyl]-O-nHex (1h): Reaction of 4 (120 mg, 0.22 mmol), thieno-
[2,3-b]pyridine-2-carbonyl chloride (44 mg, 0.24 mmol), and
Et3N (100 μL, 0.72 mmol) yielded a yellow oil (94 mg, 72%).
[R]D20: -29.62 (c 1.1, MeOH). MS (ESIþ) m/z=593 [M þ H]þ,
616 [M þ Na]þ. 1H NMR (400 MHz, CDCl3) δ 8.61 (dd, J=4.6,
1.6 Hz, 1H), 8.09 (dd, J =8.2, 1.6 Hz 1H), 8.00 (d, J =6.8 Hz,
1H), 7.78 (s, 1H), 7.32-7.22 (m, 6H), 5.88 (m, 1H), 4.65 (m, 1H),
4.15 (m, 2H), 3.46 (s, 2H), 3.23 (m, 2H), 2.81 (m, 2H), 2.41-2.17
(m, 4H), 1.89 (m, 2H), 1.65-1.55 (m, 4H), 1.39-1.24 (m, 11H),
0.85 (t, J=6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.5,
171.6, 162.2, 162.1, 148.7, 138.4, 132.7, 129.3 (2C), 128.2 (2C),
127.1, 123.0 (2C), 120.1 (2C), 65.9, 63.2, 53.5 (2C), 53.1, 37.5,
36.0, 33.3, 32.7, 31.9 (2C), 31.3, 28.4, 27.2, 25.4, 22.5, 14.0.
Purity: 99% (by HPLC). Anal. (C33H44N4O4S) C, H, N, S.
N-Thieno[2,3-b]thieno-2-yl-L-Glu[NH-2-(1-benzylpiperidin-4-
yl)ethyl]-O-nHex (1i): Reaction of 4 (144 mg, 0.26 mmol),
thieno[2,3-b]thiophene-2-carbonyl chloride (54 mg, 0.29 mmol),
and Et3N (81 μL, 0.58 mmol) afforded a yellow solid (86 mg,
55%). Mp: 100-102 °C. [R]D20: -25.73 (c 1, MeOH). MS (ESIþ)
m/z=598 [M þ H]þ. 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H),
7.68 (d, J=7.1 Hz, 1H), 7.36 (d, J=5.5 Hz, 1H), 7.29 (m, 5H),
7.23 (d, J = 5.5 Hz, 1H), 5.99 (m, 1H), 4.65 (m, 1H), 4.14 (m,
2H), 3.5 (s, 2H), 3.23 (m, 2H), 2.90 (m, 2H), 2.39-2.16 (m, 4H),
2.00 (m, 2H), 1.65-1.59 (m, 4H), 1.39-1.24 (m, 11H), 0.85 (t,
J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.5, 171.8,
162.3, 146.3, 141.6, 141.3, 129.5, 129.0 (2C), 128.3 (3C), 127.4,
120.9, 125.5, 65.9, 63.0, 53.4 (2C), 53.0, 37.4, 36.0, 33.1, 32.8,
31.5 (2C), 31.3, 28.4, 27.5, 25.4, 22.5, 14.0. Purity: 98% (by
HPLC). Anal. (C32H43N3O4S2) C, H, N, S.
N-Thieno-2-yl-L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl]-O-nHex
(1e): Reaction of 4 (148 mg, 0.27 mmol), thiophene-2-carbonyl
chloride (42 mg, 0.29 mmol), and Et3N (83 μL, 0.59 mmol)
20
provided a yellow solid (140 mg, 95%). Mp: 48-50 °C. [R]D
:
1
-22.81 (c 1.2, MeOH). MS (ESIþ) m/z = 542 [M þ H]þ. H
NMR (400 MHz, MeOD) δ 7.85 (dd, J=3.9, 1.2 Hz, 1H), 7.67
(dd, J=5.1, 1.2 Hz, 1H), 7.43 (s, 5H), 7.13 (dd, J=5.1, 3.9 Hz,
1H), 4.50 (m, 1H), 4.14 (m, 2H), 4.13 (s, 2H), 3.34-3.29 (m, 3H),
3.16 (m, 2H), 2.74 (m, 2H), 2.35 (m, 2H), 2.28 (m, 1H), 2.06 (m,
1H), 1.87 (m, 2H), 1.64 (m, 2H), 1.42 (m, 2H), 1.34-1.26 (m,
8H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, MeOD) δ
174.6, 173.3, 164.5, 139.6 (2C), 132.2, 132.0 (2C), 130.5, 130.2,
130.1 (2C), 128.9, 66.5, 62.2, 54.0 (2C), 53.7 (2C), 37.4, 36.2,
33.2, 32.5 (2C), 30.7, 29.6, 27.8, 26.7, 23.6, 14.3. Purity: 99% (by
HPLC). Anal. (C30H43N3O4S) C, H, N, S.
N-Cbz-L-Glu(OtBu)-O-nHex (5): Derivative 5 was obtained
from Cbz-N-L-Glu(OtBu)-OH (1.0 g, 2.99 mmol), PyBOP (1.7 g,
3.29 mmol), n-hexanol (367 mg, 3.59 mmol), and Et3N (830 μL,
5.99 mmol) following the method described previously to obtain
2. Then, it was purified by flash column chromatography eluting
with Hex/AcOEt (6:1) affording a colorless oil (72%). MS
1
(ESIþ) m/z = 444 [M þ H]þ. H NMR (300 MHz, CDCl3) δ
7.32 (s, 5H), 5.28 (bs, 1H), 5.00 (s, 2H), 4.31 (m, 1H), 4.05 (m,
2H), 2.19-2.40 (m, 2H), 2.01-2.17 (m, 1H), 1.82-1.93 (m, 1H),
1.57 (m, 2H), 1.40 (s, 9H), 1.12-1.28 (m, 6H), 0.80 (t, J=7.0 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 171.8, 171.7, 155.7, 136.0,
128.3 (2C), 128.0, 127.9 (2C), 80.6, 66.8, 65.6, 53.3, 31.2, 31.1,
28.2, 27.8 (3C), 27.5, 25.2, 22.2, 13.8. Purity: g99% (by HPLC).
Anal. (C23H35NO6) C, H, N.
N-4-Bromothieno-2-yl-L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl]-
O-nHex (1f): Reaction of 4 (101 mg, 0.4 mmol), 5-bromothio-
phene-2-carbonyl chloride (222 mg, 0.49 mmol), and Et3N
(124 μL, 0.89 mmol) provided a yellow oil (62 mg, 25%).
[R]D20: -28.84 (c 1, MeOH). MS (ESIþ) m/z = 622, 620 [M þ
H]þ. 1H NMR (400 MHz, MeOD) δ 7.74 (d, J = 1.4 Hz, 1H),
7.68 (d, J=1.4 Hz, 1H), 7.36 (s, 5H), 4.48 (m, 1H), 4.14 (m, 2H),
3.73 (s, 2H), 3.30 (m, 2H), 3.13 (m, 2H,), 2.40-2.36 (m, 5H), 2.32
(m, 1H), 2.07 (m, 1H), 1.77-1.74 (m, 2H), 1.40 (m, 2H), 1.32 (m,
2H), 1.31-1.27 (m, 8H), 0.87 (t, J=7.0 Hz, 3H). 13C NMR (100
MHz, MeOD) δ 174.6, 173.1, 164.5, 141.0 (2C), 132.2, 131.4
(2C), 129.9, 129.6, 129.3 (2C), 111.0, 66.6, 63.5, 54.2, 54.1, 53.8
(2C), 37.7, 36.6, 33.5, 32.5 (2C), 31.8, 29.6, 27.8, 26.7, 23.6, 14.3.
Purity: 98% (by HPLC). Anal. (C30H42BrN3O4S) C, H, N, S.
N-Benzo[b]thieno-2-yl-L-Glu[NH-2-(1-benzylpiperidin-4-yl)-
ethyl]-O-nHex (1g): Reaction of 4 (143 mg, 0.26 mmol), benzo-
[b]thiophene-2-carbonyl chloride (57 mg, 0.28 mmol), and Et3N
(83 μL, 0.57 mmol) provided a yellow solid (52 mg, 34%). Mp:
101-103 °C. [R]D20: -30.05 (c 0.9, MeOH). MS (ESIþ) m/z =
N-Cbz-L-Glu(OH)-O-nHex (6): Following the method de-
scribed to obtain 4, compound 6 was obtained from 5 (952 mg,
2.25 mmol) and TFA (2.48 mL). Then, it was purified by silica flash
column chromatography eluting with Hex/AcOEt (6:1), obtaining
a colorless solid (732 mg, 88%). Mp: 48-50 °C. MS (ESIþ) m/z=
366 [M þ H]þ. 1H NMR (400 MHz, CDCl3) δ 10.80 (s, 1H), 7.30
(s, 5H), 5.66 (m, 1H), 5.08 (s, 2H), 4.39 (m, 1H), 4.10 (m, 2H), 2.42
(m, 2H), 2.18 (m, 1H), 1.95 (m, 1H), 1.61 (m, 2H), 1.32-1.22 (m,
6H), 0.87 (t, J = 5.5 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 177.8, 172.0, 156.0, 136.0, 128.4 (2C), 128.0 (2C), 127.9, 67.0,
65.8, 53.1, 31.2, 29.8, 28.2, 27.3, 25.3, 22.3, 13.8. Purity: 99%
(by HPLC). Anal. (C19H27NO6) C, H, N.
N-Cbz-L-Glu[NH-2-(1-benzylpiperidin-4-il)ethyl]-O-nHex (1b):
Starting from 6 (109 mg, 0.30 mmol), N-benzyl-4-(2-amino-
ethyl)piperidine (60 μL, 0.27 mmol), EDC (58 mg, 0.30 mmol),
HOBt (46 mg, 0.30 mmol), Et3N (112 μL, 0.81 mmol) and
DMAP (1 mg, 0.03 mmol) and following the method described
previously to obtain 1a, derivative 1b was synthesized and then
purified by silica gel column chromatography eluting with
CH2Cl2/MeOH (95:5) to afford a colorless solid (265 mg,
65%). Mp: 88-90 °C. [R]D20: -30.02 (c 1, MeOH). MS (ESIþ)
m/z = 566 [M þ H]þ. 1H NMR (300 MHz, MeOD) δ 7.43
1
592 [M þ H]þ. H NMR (400 MHz, CDCl3) δ 7.84-7.81 (m,
2H), 7.76 (d, J=7.1 Hz, 1H), 7.39-7.35 (m, 3H), 7.27 (m, 5H),
4.66-4.64 (m, 1H), 4.14 (m, 2H), 3.5 (s, 2H), 3.25-3.17 (m, 2H),
2.83 (m, 2H), 2.40-2.14 (m, 4H), 1.92 (m, 2H), 1.64-1.53 (m,
4H), 1.39-1.22 (m, 11H), 0.85 (t, 3H). 13C NMR (100 MHz,
CDCl3) δ 172.6, 171.0, 162.0, 141.4 (2C), 139.4, 138.3, 129.6

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7255
(m, 10H), 5.10 (s, 2H), 4.50 (m, 1H), 4.14 (m, 2H), 4.13 (s, 2H),
3.34-3.29 (m, 3H), 3.16 (m, 2H), 2.74 (m, 2H), 2.35 (m, 2H),
2.28 (m, 1H), 2.06 (m, 1H), 1.87 (m, 2H), 1.64 (m, 2H), 1.42 (m,
2H), 1.34-1.26 (m, 8H), 0.86 (t, J = 5.5 Hz, 3H). 13C NMR
(75 MHz, MeOD) δ 174.6, 173.3, 164.5, 139.6 (2C), 132.2 (2C),
132.0 (2C), 130.5, 130.2, 130.1 (2C), 128.9 (2C), 66.5, 65.3, 62.2,
54.0 (2C), 53.7 (2C), 37.4, 36.2, 33.2, 32.5 (2C), 30.7, 29.6, 27.8,
26.7, 23.6, 14.3. Purity: 99% (by HPLC). Anal. (C33H47N3O5)
C, H, N.
Biochemical Methods. In Vitro Cholinesterase Inhibition As-
says. AChE from bovine erythrocytes (0.25-1.0 unit/mg, ly-
ophilized powder), AChE from human erythrocytes (min.
500 units/mg protein in buffered aqueous solution), BuChE
from equine serum (10 units/mg protein, lyophilized powder),
and BuChE from human serum (3 units/mg protein, lyophilized
powder) were purchased from Sigma. Compounds were evalu-
ated in 100 mM phosphate buffer, pH 8.0, at 30 °C using
acetylthiocholine and butyrylthiocholine (0.4 mM) as sub-
strates, respectively. In both cases, 5,50-dithio-bis(2-nitroben-
zoic)acid (DTNB, Ellman0s reagent, 0.2 mM) was used, and the
values of IC50 were calculated by UV spectroscopy from the
absorbance changes at 412 nm. Experiments were performed in
triplicate.
In Vitro Blood-Brain Barrier Permeation Assay. Prediction
of the brain penetration was evaluated using a parallel artificial
membrane permeation assay (PAMPA), in a similar manner as
described previously.37 Commercial drugs, phosphate-buffered
saline solution at pH 7.4 (PBS), and dodecane were purchased
from Sigma, Aldrich, Acros, and Fluka. Millex filter units
(PVDF membrane, diameter 25 mm, pore size 0.45 μm) were
acquired from Millipore. The porcine brain lipid (PBL) was
obtained from Avanti Polar Lipids. The donor microplate was a
96-well filter plate (PVDF membrane, pore size 0.45 μm), and
the acceptor microplate was an indented 96-well plate, both
from Millipore. The acceptor 96-well microplate was filled with
180 μL of PBS/ethanol (7:3), and the filter surface of the donor
microplate was impregnated with 4 μL of porcine brain lipid
(PBL) in dodecane (20 mg mL-1). Compounds were dissolved in
PBS/ethanol (7:3) at 1 mg mL-1, filtered through a Millex filter,
and then added to the donor wells (180 μL). The donor filter
plate was carefully put on the acceptor plate to form a sandwich,
which was left undisturbed for 4 h at 25 °C. After incubation, the
donor plate is carefully removed, and the concentration of
compounds in the acceptor wells was determined by UV spec-
troscopy. Every sample is analyzed at five wavelengths, in four
wells, and at least in three independent runs, and the results are
given as the mean ( standard deviation. In each experiment,
15 quality control standards of known BBB permeability were
included to validate the analysis set.
Measurement of Propidium Iodide Displacement from the
Peripheral Anionic Site (PAS) of AChE. A solution of AChE
from bovine erythrocytes at a concentration of 5 μM in 0.1 mM
Tris buffer, pH 8.0, was used. Aliquots of compounds to get the
final concentrations of 0.3, 1.0, and 3.0 μM were added, and the
solutions were kept at room temperature for at least 6 h. After,
the samples were incubated for 15 min with propidium iodide at
a final concentration of 20 μM, and the fluorescence was
measured in a fluorescence microplate reader (Fluostar Optima,
BMG, Germany). Wavelengths of excitation and emission were
485 and 620 nm, respectively.
subcultured in 48 well plates at a seeding density of 105 cells per
well. For the cytotoxicity experiments, cells were treated with
drugs before confluence in DMEM free of serum.
To study the cytotoxic effects of the compounds alone, cells
were plated at a density of 105 cells per well at least 48 h before
the toxicity measurements. Cells were exposed for 24 h to the
compound at 30 μM, and the quantitative assessment of cell
death was made by measurement of the percent of the intra-
cellular enzyme lactate dehydrogenase (LDH) released to the
extracellular medium (cytotoxicity detection kit, Roche). The
quantity of LDH was evaluated in a microplate reader (Anthos
2010 or Labsystems iMES Reader MS) at 492 nm (λ excitation)
and 620 nm (λ emission).
To study the cytoprotective action of the compounds against
cell death induced by 60 μM H2O2 or the mixture of 30 μM
rotenone and 10 μM oligomycin A, drugs were given at time zero
and maintained for 24 h. Then, the media were replaced by fresh
media still containing the drug plus the cytotoxic insult, which
was left for an additional 24 h period. Thereafter, cell survival
was assessed measuring LDH activity. When the combination of
rotenone (30 μM) plus oligomycin A (10 μM) was used as toxic
insult, two protocols were used: one was exactly the same as the
one used for H2O2 (the preincubation protocol), and the second
procedure, termed co-incubation protocol, consisted of incubat-
ing the drugs only when the toxics were present.
Measurement of Lactic Dehydrogenase (LDH) Activity. Ex-
tracellular and intracellular LDH activity was measured by
UV-vis using a cytotoxicity cell death kit (Roche-Boehringer.
Mannheim, Germany) according to the manufacturer’s indica-
tions. Total LDH activity was defined as the sum of intracellular
and extracellular LDH activity and released LDH was defined
as the percentage of extracellular compared with total LDH
activity. Data were expressed as the mean ( SEM of at least
three different cultures in quadruplicate. LDH released was
calculated for each individual experiment considering 100%
the extracellular LDH released by the vehicle with respect to
the total. To determinate percent protection, LDH release was
normalized as follows: in each individual triplicate experiment,
LDH release obtained in nontreated cells (basal) was subtracted
from the LDH released upon the toxic treatment and normal-
ized to 100% and that value was subtracted from 100.
Acknowledgment. The authors gratefully acknowledge the
financial support of Spanish Ministry of Science and Innova-
tion (projects SAF 2006-01249, SAF 2006-08540, and SAF-
2006-03589), Comunidad de Madrid (Programa de Activi-
ꢀ
dades de IþD entre Grupos de Investigacion en Biociencias,
project S-SAL/0275/2006), and the Spanish Ministry of
Health (Instituto de Salud Carlos III) RETICS-RD06/0026,
and Agencia Laın Entralgo. The fellowships to M.P.A. and
´
G.C.G.-M. from MEC and CSIC, respectively, are also
acknowledged.
Supporting Information Available: Elemental analysis of new
derivatives. This material is available free of charge via the
Internet at http://pubs.acs.org.
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