New melatonin- N, N -dibenzyl(N -methyl)amine hybrids: Potent neurogenic agents with antioxidant, cholinergic, and neuroprotective properties as innovative drugs for alzheimers disease

Article abstract of DOI:10.1021/jm5000613

Here, we describe a new family of melatonin-N,N-dibenzyl(N-methyl)amine hybrids that show a balanced multifunctional profile covering neurogenic, antioxidant, cholinergic, and neuroprotective properties at low-micromolar concentrations. They promote maturation of neural stem cells into a neuronal phenotype and thus they could contribute to CNS repair. They also protect neural cells against mitochondrial oxidative stress, show antioxidant properties, and inhibit human acetylcholinesterase (AChE). Moreover, they displace propidium from the peripheral anionic site of AChE, preventing the β-amyloid aggregation promoted by AChE. In addition, they show low cell toxicity and can penetrate into the CNS. This multifunctional profile highlights these melatonin-N,N-dibenzyl(N-methyl)amine hybrids as useful prototypes in the research of innovative drugs for Alzheimers disease.

Full text of DOI:10.1021/jm5000613

Article
pubs.acs.org/jmc
New Melatonin−N,N‑Dibenzyl(N‑methyl)amine Hybrids: Potent
Neurogenic Agents with Antioxidant, Cholinergic, and
Neuroprotective Properties as Innovative Drugs for Alzheimer’s
Disease
Beatriz Lopez-Iglesias,† Concepcion Perez,† Jose A. Morales-García,‡,§ Sandra Alonso-Gil,‡,§
́
́
́
́
Ana Perez-Castillo,‡,§ Alejandro Romero,∥,⊥ Manuela G. Lopez,∥ Mercedes Villarroya,∥ Santiago Conde,†
́
́
and María Isabel Rodríguez-Franco*,†
†Instituto de Química Med
Spain
‡Instituto de Investigaciones Biomed
Duperier 4, 28029 Madrid, Spain
§Centro de Investigacion
Biomed
Madrid, Spain
∥Instituto Teofi
́
ica, Consejo Superior de Investigaciones Científicas (IQM-CSIC), C/Juan de la Cierva 3, 28006 Madrid,
́
icas “Alberto Sols”, Consejo Superior de Investigaciones Científicas (IIB-CSIC), C/Arturo
́
́
ica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), C/ Valderrebollo 5, 28031
́
́ ́
lo Hernando and Departamento de Farmacología y Terapeutica, Facultad de Medicina, Universidad Autonoma de
Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
S
* Supporting Information
ABSTRACT: Here, we describe a new family of melatonin−N,N-dibenzyl(N-
methyl)amine hybrids that show a balanced multifunctional profile covering
neurogenic, antioxidant, cholinergic, and neuroprotective properties at low-
micromolar concentrations. They promote maturation of neural stem cells into a
neuronal phenotype and thus they could contribute to CNS repair. They also
protect neural cells against mitochondrial oxidative stress, show antioxidant
properties, and inhibit human acetylcholinesterase (AChE). Moreover, they
displace propidium from the peripheral anionic site of AChE, preventing the β-
amyloid aggregation promoted by AChE. In addition, they show low cell toxicity
and can penetrate into the CNS. This multifunctional profile highlights these melatonin−N,N-dibenzyl(N-methyl)amine hybrids
as useful prototypes in the research of innovative drugs for Alzheimer’s disease.
noncholinergic functions, including an important role in Aβ
processing. Several studies have indicated that AChE increases
the formation of Aβ fibrils in vitro5 and Aβ plaques in the
cerebral cortex of transgenic mouse models of AD.6 These
effects seem to be mediated by interactions between Aβ and the
peripheral anionic site (PAS) of the enzyme,7 which is
supported by the fact that propidium, a noncompetitive
AChE inhibitor that binds purely to PAS, significantly decreases
AChE-induced Aβ aggregation; however, edrophonium, a pure
competitive inhibitor that binds the center active site (CAS),
does not show any effect on AChE-induced Aβ fibrillogenesis.8
These findings have led to the design and synthesis of inhibitors
of both the CAS and PAS for use as promising disease-
modifying AD drug candidates because they can simultaneously
improve cognition and slow the rate of Aβ degeneration.9
Recently, this hypothesis has been validated in murine models
of AD that showed an improvement in cognition and a
INTRODUCTION
■
Alzheimer’s disease (AD) is a dreadful neurological illness and
is the most frequent of the degenerative dementias. The slow
but progressive impairment of the physical and neurological
conditions of AD patients produces devastating effects on the
patients themselves and on their caregivers as well as a very
high economic burden for families and public health systems.1
AD shows a highly complex network of interconnected
pathological processes that result in the accumulation of
abnormal deposits of β-amyloid peptide (Aβ) and hyper-
phosphorylated tau protein as well as massive cell death and the
loss of synapses, especially in the cholinergic system.2
Currently, the therapeutic options for the treatment of AD
are limited to three acetylcholinesterase (AChE) inhibitors,
namely, donepezil, rivastigmine, and galantamine,3 and one N-
methyl-D-aspartate receptor antagonist, memantine.4
The main role of AChE is to hydrolyze the neurotransmitter
acetylcholine (ACh) after the occurrence of a nerve impulse.
Thus, inhibition of this enzyme has been largely used as a
rational approach to palliate memory deficits by increasing ACh
in the synaptic cleft. However, AChE also plays other
Received: January 13, 2014
Published: April 16, 2014
© 2014 American Chemical Society
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reduction of brain amyloid burden when mice were treated with
dual-binding-site AChE inhibitors.10−13
no peripheral MAO inhibitory effects in in vivo experiments.35
Huprine X is a subnanomolar AChE inhibitor that possesses
additional effects, such as inhibition of BACE-1, activation of α-
secretases, and stimulation of protein kinase C/mitogen-
activated protein kinase pathway signaling, and was shown to
improve learning and memory in a triple transgenic mouse
model of AD.36,37 Our group has also reported several families
of MTLs that combined neuroprotective, cholinergic, and
antioxidant properties,38−40 including melatonin-based hybrids
that have proved their effectiveness in a murine model of
AD.11,41,42
Continuing with our interest in the design, synthesis, and
biological evaluation of multifunctional molecules, our work is
currently focused on taking advantage of the potential
neurogenic profile of melatonin-based hybrids, which are
endowed with additional anticholinergic properties. Thus, in
this work, we have designed new melatonin− N,N-dibenzyl(N-
methyl)amine hybrids (1−14) by binding two fragments with
interesting and complementary properties. The melatonin
framework, in addition to its above-mentioned neurogenic
profile, could demonstrate antioxidant and neuroprotective
features and could also interact with the AChE-PAS because of
Because of the progressive failure of the endogenous
antioxidant systems, the oxidative damage in cellular structures
is augmented during aging, and recent findings have shown the
involvement of oxidative imbalance in different pathologies. For
instance, a post-mortem study of the frontal cortex of AD
patients demonstrated a significant disease-dependent increase
in oxidative markers that correlated with mini-mental status
examination scores.14 Additionally, in a murine AD model,
oxidative damage was found to be an event that precedes the
appearance of other pathological hallmarks of AD.15 These
findings pointed out the early involvement of mitochondrial
oxidative stress in the pathogenesis and progression of this
disease.16 Thus, drugs that protect neurons from mitochondrial
oxidative stress could be useful either for the prevention or
treatment of AD.17
Neurogenesis is a dynamic process occurring in the brain of
adult vertebrates whereby new nervous cells are generated
along the lifespan of an individual. Although it was thought for
many years that the creation of nervous tissue was restricted to
the first stages of embryonic development, this concept
changed in 1962 when Altman demonstrated the formation
of new neurons in the brain of adult rats.18 In the following
years, neurogenesis was also found in different species of
vertebrates, including humans.19,20 The whole process com-
prises several stages, from the proliferation of progenitor cells
to the integration of these new cells into cerebral circuits.
Because renewal rates for neuronal cells decrease with aging,
drugs capable of helping the replacement of damaged cells with
healthy ones would represent an exciting new therapeutic
treatment for AD and other neurodegenerative diseases.21
Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous
molecule produced by a variety of organs and tissues. It is
involved in many physiological processes, such as circadian
rhythm, endogenous antioxidant regulation, and the immune
system. The natural decline in melatonin levels with aging has
been associated with insomnia and depression as well as the
development of neurodegenerative disorders such as AD.22
Among its extensive pharmacological profile, melatonin shows a
potent antioxidant activity by the direct capture of free
radicals,23 stimulates the synthesis of several endogenous
antioxidant enzymes,24 improves mitochondrial energy metab-
olism,25 decreases neurofilament hyperphosphorylation, and
plays a neuroprotective role against Aβ.26 Moreover, melatonin
potentiates the proliferation and differentiation of neural stem
cells in the hippocampus of adult mice.27−29 Thus, melatonin-
based compounds could show an interesting neurogenic profile
of paramount importance in the search of new therapeutics for
AD and other neurodegenerative diseases.
its aromatic character, as we observed in the tacrine−melatonin
42
series.
The second selected fragment was the protonable
N,N-dibenzyl(N-methyl)amine, which is present in the well-
known AChE inhibitor AP2238, because its interaction with the
AChE-CAS has been probed43−45 (Figure 1).
Figure 1. Structures of melatonin, AP2238, and new melatonin− N,N-
dibenzyl(N-methyl)amine hybrids (1−14).
In this article, we describe the synthesis of new melatonin−
N,N-dibenzyl(N-methyl)amine hybrids (1−14) and their
biological evaluation, which comprises inhibition of human
AChE and BuChE, displacement of propidium from AChE-
PAS, in vitro CNS permeation, and oxygen radical absorbance
capacity (ORAC). Using a human neuroblastoma cell line, we
also studied the cell viability and the neuroprotective effects of
these hybrids against death induced by mitochondrial oxidative
stress. Lastly, we explored the neurogenic effects of a selection
of these compounds using primary hippocampal neural stem
cells from adult rats.
Furthermore, the complexity of AD pathology has
encouraged the design of molecules that are able to interact
with two or more complementary targets, with the expectation
that these multitarget ligands (MTL) may represent important
advantages in the treatment of the disease.30−32 In recent years,
many interesting MTLs have been developed, such as
memoquin, ladostigil, and huprine X, among others. Memoquin
is an AChE and β-secretase-1 (BACE-1) inhibitor with
additional antioxidant and Aβ anti-aggregating properties,
which has recently been proven to have in vivo efficacy using
different mouse models of induced amnesia.33,34 Ladostigil is an
inhibitor of cholinesterases (ChEs) and is a brain-selective
inhibitor of monoaminoxidases (MAO-A and -B), with little or
RESULTS AND DISCUSSION
■
Synthesis of Melatonin−N, N-Dibenzyl(N-methyl)-
amine Hybrids . New melatonin−N,N-dibenzyl(N-methyl)-
amine hybrids 1−14 were obtained as depicted in Scheme 1.
Reaction of commercially available 4-(bromomethyl)-
benzonitrile and differently substituted N-methylbenzylamines
gave 4-{[benzyl(methyl)amino]methyl}benzonitriles 15 46−19
(77−93% yield), which were hydrolyzed to corresponding acid
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Scheme 1. Synthesis of Melatonin−N,N-Dibenzyl(N-methyl)amine Hybrids (1−14)
Table 1. Yield (%), Inhibition of h-AChE and h-BuChE [IC50 (μM) or Percentage of Inhibition at 10 μM (%)], and
Displacement of Propidium Iodide from the Peripheral Anionic Site of AChE (%) by New Melatonin−N,N-Dibenzyl(N-
methyl)amine Hybrids 1−14
b
b
IC50 (μM) or (inhibition % at 10 μM)
displacement of propidium iodide (%)
a
R
R′
yield (%)
h-AChE
h-BuChE
3.7 0.1
0.3 μM
1.0 μM
3.0 μM
1
H
H
H
H
H
H
46
30
72
65
65
49
57
55
55
82
61
76
78
63
1.9 0.1
4.2 0.2
6.1 0.4
4.1 0.3
2.5 0.1
3.8 0.2
2.6 0.1
5.1 0.2
5.6 0.4
2.1 0.1
4.7 0.2
5.4 0.2
3.8 0.1
6.8 0.2
0.23 0.07
n.d.
37.6 0.22
8.0 0.54
45.2 1.80
24.1 1.01
8.1 0.87
7.7 0.08
n.d.
5.6 0.11
22.4 0.37
25.3 0.96
24.9 0.30
n.d.
51.4 1.80
25.3 0.70
67.3 0.08
17.8 0.31
34.5 0.29
34.7 0.61
n.d.
2
5-OH
5-OCH3
6-OCH3
6-F
9.5 0.5
7.8 0.5
50.0 2.1
7.0 0.1
>10 (38%)
>10 (37%)
>10 (38%)
>10 (38%)
>10 (22%)
8.2 0.4
>10 (47%)
9.6 0.2
>10 (45%)
0.040 0.002
n.d.
3
4
5
6
2-Cl
H
25.8 0.90
n.d.
7
2-Cl
5-OCH3
H
8
3-Cl
10.9 0.30
4.1 0.70
16.8 0.06
11.5 0.32
n.d.
15.6 0.32
11.3 0.48
11.4 0.01
16.9 1.21
n.d.
29.9 0.60
31.8 0.37
33.4 0.38
37.9 1.23
30.4 0.76
43.7 1.10
n.d.
9
3-Cl
5-OCH3
6-OCH3
H
10
3-Cl
11
2-OCH3
2-OCH3
3-OCH3
3-OCH3
12
5-OCH3
H
13
21.8 0.56
n.d.
21.4 1.18
n.d.
14
5-OCH3
tacrine
BW284c51
n.d.
n.d.
n.d.
n.d.
n.d.
16.0 2.4
a
b
Percentage of isolated product (%). Results are the mean SEM (n = 3). n.d., not determined.
derivatives 20−24 in good yields (69−97%). Then, these
intermediate acids were activated with benzotriazol-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and
subsequently coupled with commercially available tryptamines
in the presence of triethylamine in dimethylformamide
solutions at room temperature overnight to afford the desired
melatonin− N,N-dibenzyl(N-methyl)amine derivatives (1−14)
in moderate to good yields (30−82%). All new hybrids (1−14)
were purified by chromatographic techniques and structurally
characterized by their analytical (HPLC and combustion
analysis) and spectroscopic data (1H NMR, 13C NMR, and
MS).
(N-methyl)amine hybrids 1−14 were assayed as inhibitors of
human AChE (h-AChE) and BuChE (h-BuChE) by following
the method of Ellman et al.,47 using tacrine as a reference
(Table 1). All tested compounds displayed inhibition of h-
AChE with IC50’s in the low-micromolar range and showed
little fluctuation after the introduction of different substituents
in the benzene or indole ring. However, h-BuChE inhibition
showed a higher dependency on the presence of substituents:
only unsubstituted hybrid 1 displayed low-micromolar
inhibition of h-BuChE (IC50 = 3.7 μM), whereas the
introduction of any substituent in 2−14 penalized the
enzyme−hybrid interactions by 1 order of magnitude and
showed IC50’s around 10 μM or higher.
Inhibition of Human Cholinesterases and Propidium
Displacement from AChE. New melatonin−N, N-dibenzyl-
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As previously mentioned, the enzyme AChE interacts with
Aβ oligomers and stimulates conformational changes that
contribute to their aggregation and precipitation into amyloid
plaques.7 The enzymatic region that promotes these abnormal
modifications is placed in the PAS of AChE48 because the
selective PAS ligand propidium inhibits AChE-induced Aβ
aggregation, whereas the competitive CAS ligand edrophonium
does not.8 Therefore, the propidium displacement assay is a
well-accepted method and is usually used as a first approach to
study the inhibition of Aβ aggregation by PAS ligands.49
Propidium iodide undergoes a 10-fold increase in fluo-
rescence when it is bound to AChE-PAS. In the presence of
another inhibitor, the decay in the fluorescence signal is related
to the displacement percentage of propidium from this
enzymatic site and thus it is accepted as a measure of the
affinity of such a compound for AChE-PAS.50 Thus, the
experimental affinity of new compounds 1−14 for AChE-PAS
was studied by competitive enzymatic assays in the presence of
propidium iodide as a first and indirect probe of the inhibition
of Aβ aggregation.
Table 2. Permeability Values from the PAMPA-BBB Assay
(Pe, 10−6 cm s−1) and Oxygen Radical Absorbance Capacity
by Fluorescence (ORAC-FL, Trolox Equiv) of Hybrids 1−14
PAMPA-BBB assay
ORAC-FL
a
a
compd
R
R′
Pe (10−6 cm s−1
)
(trolox equiv)
1
H
H
H
H
H
H
7.4 0.2
5.0 0.5
7.1 0.4
7.2 0.1
6.6 0.2
5.8 0.1
5.9 0.4
7.1 0.1
7.9 0.2
7.9 0.1
8.8 0.3
9.9 0.3
8.8 0.1
8.7 0.4
n.d.
3.2 0.03
4.3 0.1
2.5 0.04
1.5 0.01
2.1 0.04
2.8 0.02
2.5 0.2
2.9 0.1
2.6 0.1
1.8 0.04
2.9 0.1
2.4 0.1
2.7 0.04
2.5 0.04
2.4 0.1
2
5-OH
5-OCH3
6-OCH3
6-F
3
4
5
6
2-Cl
H
7
2-Cl
5-OCH3
H
8
3-Cl
9
3-Cl
5-OCH3
6-OCH3
H
10
11
12
13
14
3-Cl
2-OCH3
2-OCH3
3-OCH3
3-OCH3
5-OCH3
H
5-OCH3
melatonin
New hybrids 1−14 were evaluated at three concentrations,
0.3, 1.0, and 3.0 μM, and the results are gathered in Table 1. All
compounds displaced the propidium cation from the AChE-
PAS better than 4,4′-(3-oxopentane-1,5-diyl)bis(N,N-dimethyl-
N-prop-2-en-1-ylanilinium) dibromide (BW248c51), a PAS
ligand used as a reference in this type of experiment.40 These
results suggested that the new hybrids were able to bind to
AChE-PAS and thus they could prevent Aβ aggregation
stimulated by this enzyme. For the majority of compounds, a
concentration−response relationship was obtained, yielding a
maximum at 3.0 μM, which is a concentration that is very close
to the IC50 values observed for h-AChE inhibition (1.9−6.8
μM). However, some compounds (1, 3, and 10) displayed a U-
shaped line in the concentration−response data, as observed in
other MTLs in a previous work.40 This could be explained by
the fact that these multifunctional derivatives could interact
with other points of the enzymatic gorge, depending on their
concentration.
a
Results are the mean SD (n = 3).
of new melatonin− N,N-dibenzyl(N-methyl)amine hybrids 1−
14 was determined as a measure of their antioxidant properties
(Table 2) following a well-established protocol.52 Trolox, the
aromatic part of vitamin E and the part that is responsible for
radical capturing, was used as an internal standard to which the
unit value is arbitrarily given, ORAC (trolox) = 1. Thus, results
were expressed as trolox equivalents (micromoles of trolox/
micromoles of tested compound) on a comparative scale that
shows if the products are better (ORAC > 1) or worse than
trolox (ORAC < 1). Melatonin was used as a positive standard
for comparative purposes, giving an ORAC value of 2.4 that
fully agreed with the value previously described by Sofic et al.
(2.0 trolox equiv).
peroxyl radical absorbance capacities ranging from 1.5- to 4.3-
fold of the trolox value and thus they could be considered as
potent antioxidant agents.
As expected, the ORAC of these compounds is mainly
located in the melatonin-like fragment, where the presence of a
phenolic group clearly yielded the best ORAC value (2, R′ = 5-
OH, ORAC = 4.3 trolox equiv). However, the introduction of a
halogen or a methoxy group in the indole and/or the N,N-
dibenzyl(N-methyl)amine fragment penalized the ORAC
ability to between 0.3 and 1.7 trolox equiv compared to
unsubstituted hybrid 1 (ORAC = 3.2 trolox equiv). It is worth
mentioning that 5-methoxyindole derivatives 3 and 9 showed
better ORAC values (2.5 and 2.6 trolox equiv, respectively)
than their 6-methoxy counterparts 4 and 10 (1.5 and 1.8 trolox
equiv, respectively), suggesting the involvement of the indolic
position 5 in the trapping mechanism of these hybrids.
Cell Viability and Neuroprotection Studies. To explore
the therapeutic potential of new melatonin−dibenzylamine
hybrids 1−14, cell viability and neuroprotective capacity against
mitochondrial oxidative stress were evaluated using the human
neuroblastoma cell line SH-SY5Y with a mixture of rotenone
and oligomycin A as the toxic insult. The combination of such
toxic agents induces mitochondrial ROS as a consequence of
the blockade of complexes I and V of the mitochondrial
electronic chain; thus, it is a good model of endogenous
oxidative stress.54 In these experiments, compounds were tested
at 1.0 μM, a concentration close to their IC50 in h-AChE
53
Tested compounds showed potent
At 3.0 μM, hybrid 3, which is derived from 5-methoxyindole
and an unsubstituted dibenzylamine fragment, showed the best
result with 67% propidium displacement. Other outstanding
compounds were 1 (51%), 11 (38%), and 13 (44%).
In Vitro Blood−Brain Barrier Permeation Assay. To
explore whether new hybrids 1−14 could enter into the brain,
we used a parallel artificial membrane permeation assay for the
blood−brain barrier (PAMPA-BBB), described by Di et al.,51
and then optimized these for molecules with limited water
solubility.38,41 The in vitro permeabilities (Pe) of new
compounds 1−14 and 10 commercial drugs through a lipid
extract of porcine brain were determined using a mixture of
PBS/EtOH (70:30). Assay validation was performed by
comparing the experimental permeability with the reported
values of these commercial drugs, which gave a good linear
correlation, Pe (exptl) = 0.57 Pe (bibl) + 2.36 (r2 = 0.919) (see
the Supporting Information). From this equation and taking
into account the described limits for BBB permeation,51 we
found that compounds with permeability values above 4.6 ×
10−6 cm s−1 could penetrate into the CNS by passive diffusion.
All melatonin− N,N-dibenzyl(N-methyl)amine hybrids 1−14
showed permeability values over this limit, pointing out that
these compounds could cross the BBB (Table 2).
Evaluation of the Oxygen Radical Absorbance
Capacity. The oxygen radical absorbance capacity (ORAC)
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inhibition. The percentage of cell death was determined by
measuring the amount of lactic dehydrogenase (LDH), an
enzyme that is released to the extracellular medium when
neurons die.55
First, possible cytotoxic effects of 1−14 were studied by
exposing cells to compounds at 1.0 μM for 24 h. In all cases,
the LDH percentage was lower or equal to the basal value,
suggesting that cell viability reached 100% (data not shown).
Then, cells were incubated with hybrids 1−14 at 1.0 μM for 24
h before the addition of a mixture of rotenone (30 μM) and
oligomycin A (10 μM) and then were maintained for another
24 h. Melatonin was used as a positive reference, and results are
shown in Figure 2 (and Table S3 in the Supporting
which were induced to proliferate following described
protocols.56−58 Then, we analyzed whether addition of different
compounds could promote early stages of neurogenesis and
neuronal maturation by staining the cells for β-tubulin
(antibody clone TuJ1) and MAP-2 (microtubule-associated
protein 2), respectively. All tested compounds, including
references, were used at a concentration of 10 μM, which
was determined on the basis of our experience with
neurosphere-based neurogenic experiments.57,58
Moreover, we performed additional viability studies with
compounds 3, 11, and 14 at 10 μM using the human
neuroblastoma cell line. None of tested compounds caused a
significant reduction of cell viability (measured as MTT
reduction) or cell death (measured as LDH release) after
exposing the SH-SY5Y cells for 48 h to compounds at 10 μM
(see Figure S1 and Table S4 in the Supporting Information).
These results indicated that compounds 3, 11, and 14 were not
toxic at the concentration used in the neurogenic studies.
Figures 3 and 4 show the neurogenic effects of compounds 3,
11, and 14 at 10 μM on neural stem cell cultures, which are
compared with the effect of vehicle (basal), melatonin
(endogenous substrate of melatonin receptors), and luzindole
(antagonist of melatonin receptors). In each bar chart,
quantifications of TuJ+ or MAP-2+ cells are shown using five
neurospheres per condition (Figures 3 and 4). As expected,
melatonin induced both early neurogenesis and cell maturation,
whereas luzindole was not able to promote either of these
processes. All tested melatonin− N,N-dibenzyl(N-methyl)-
amine hybrids, 3, 11, and 14, were able to stimulate early
neurogenesis and cell maturation into a neuronal phenotype
and were more effective than melatonin itself. By using TuJ1
fluorescence immunodetection, compound 11 was found to be
the most effective hybrid for early neurogenesis, being twice as
potent as melatonin (Figure 3). However, in the cell maturation
experiments, the most efficient agent was hybrid 3, derived
from 5-methoxyindole and an unsubstituted N,N-dibenzyl(N-
methyl)amine, which was almost twice as efficient as melatonin.
Moreover, hybrid 3 showed a dense crown of both β-tubulin-
and MAP-2-positive cells around the neurosphere core and
additional positive cells outside of it, suggesting a migration out
of the neurosphere (Figure 4).
Figure 2. Effect of compounds 1−14 and melatonin on cell death
induced by the combination of rotenone and oligomycin A. Human
neuroblastoma SH-SY5Y cells were exposed for 24 h to compounds
(1.0 μM) and then exposed for another 24 h to the combination of
rotenone (30 μM) and oligomycin A (10 μM) in the presence of
compounds. Cell viability was assessed by measuring LDH release.
Data are expressed as the mean SEM of triplicates of seven different
batches of cells. ***p < 0.001 with respect to toxic agents alone
(ANOVA followed by Bonferroni post-test).
Information). All new compounds displayed good levels of
protection ranging from 12 to 36%. Among hybrids with an
unsubstituted N,N-dibenzyl(N-methyl)amine fragment (1−5),
the introduction of a 5- or a 6-substituent in the indole ring
increased the protection percentage, especially in the case of
the 5-methoxy group. In fact, hybrid 3, derived from 5-
methoxyindole and an unsubstituted dibenzylamine fragment,
was the best neuroprotective agent, saving 36% of the cells from
the damage induced by mitochondrial ROS.
Regarding 5-methoxyindole derivatives (R′ = 5-OCH3), the
introduction of a chlorine atom in the N,N-dibenzyl(N-
methyl)amine fragment had a detrimental effect on neuro-
protection, reducing values from the highest value of 36% (3, R
= H) to 27% (7, R = 2-Cl) and 12% (9, R = 3-Cl). However,
the introduction of a second methoxy group in position 3
maintained an approximately equal degree of neuroprotection,
30% (14, R = 3-OCH3).
Neurogenic Studies. To evaluate the potential ability of
the new hybrids to repair brain damage, we performed in vitro
neurogenesis studies. Considering the structure of the neuro-
genic melatonin, which is a 5-methoxyindole derivative, hybrids
bearing one or two methoxy groups in different positions were
selected for these experiments, namely, 3 (R = H, R′ = 5-
OCH3), 11 (R = 2-OCH3, R′ = H), and 14 (R = 3-OCH3, R′ =
5-OCH3) (see Scheme 1 for R and R′ definitions). Primary
neural stem cells were obtained from the subgranular zone of
the dentate gyrus of the hippocampus of adult Wistar rats,
CONCLUSIONS
■
The results presented here clearly show that the new
melatonin− N,N-dibenzyl(N-methyl)amine hybrids display a
balanced biological profile. At low-micromolar concentrations,
they stimulate early stages of neurogenesis from neural stem
cells and neuronal maturation into a neuronal phenotype,
inhibit h-AChE, displace propidium from AChE-PAS, and
protect a human neuroblastoma cell line against damage caused
by mitochondrial free radicals. In addition, they are CNS-
permeable compounds with low cell toxicity that show good
antioxidant properties.
Thus, it is expected that these new melatonin−N,N-
dibenzyl(N-methyl)amine hybrids could promote autorepair
processes from neural stem cells in the CNS, protect neurons
from mitochondrial oxidative stress, increase patient memory,
and reduce the formation of amyloid plaques. These biological
properties, along with their ability to penetrate into the CNS,
highlight these new hybrids as exciting new multifunctional
prototypes in the search for new regenerative drugs for the
treatment of AD and other neurodegenerative pathologies.
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Figure 4. Neural stem cell cultures (neurospheres), derived from the
subgranular zone of the dentate gyrus of the hippocampus of adult
Wistar rats, treated with vehicle (basal), melatonin, luzindole (MT
antagonist), 3, 11, and 14 at 10 μM for 48 h. MAP-2 (red) was used as
a marker that is associated with neuronal maturation, and DAPI (blue)
was used for nuclear staining. Scale bar, 200 μm. Quantification of
MAP-2+ cells in neurospheres is shown. Values are the mean SD
from five neurospheres per condition. *** p ≤ 0.001.
Figure 3. Neural stem cell cultures (neurospheres), derived from the
subgranular zone of the dentate gyrus of the hippocampus of adult
Wistar rats, treated with vehicle (basal), melatonin, luzindole (MT
antagonist), 3, 11, and 14 at 10 μM for 48 h. Neuronal cells were
detected using an anti-β-tubulin antibody (TuJ1 clone, green) as an
early neurogenesis marker. DAPI (blue) was used for nuclear staining.
Scale bar, 200 μm. Quantification of TuJ+ cells in neurospheres is
shown. Values are the mean
SD from five neurospheres per
condition. *** p ≤ 0.001.
detector (λ = 214−274 nm) and using a Delta Pak C18 5 μm, 300 Å
column.
EXPERIMENTAL SECTION
Melting points (uncorrected) were determined with a Reichert-Jung
Thermovar apparatus. 1H and 13C NMR spectra were recorded in
CDCl3 or CD3OD solutions using a Varian XL-300 spectrometer.
Chemical shifts are reported in δ (ppm) relative to internal Me4Si. J
values are given in hertz, and spin multiplicities are expressed as s
(singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Mass
spectra (MS) were obtained by electron spray ionization (ESI) in
positive mode using a Hewlett-Packard MSD 1100 spectrometer.
■
Chemistry. General Methods. Reagents and solvents were
purchased from common commercial suppliers and were used without
further purification. Chromatographic separations were performed on
silica gel (Kielgel 60 Merck of 230−400 mesh), and compounds were
detected with UV light (λ = 254 nm). HPLC analyses were used to
confirm the purity of all compounds (>95%) and were performed on
Waters 6000 equipment at a flow rate of 1.0 mL/min with a UV
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Elemental analyses were carried out in a PerkinElmer 240C in the
N-Benzyl-1-(4-carboxyphenyl)-N-methylmethanaminium
Chloride (20). From 4-{[benzyl(methyl)amino]methyl}benzonitrile
(15) (898 mg, 3.8 mmol), 727 mg (75%) of 20 was obtained as a pure
́
Centro de Quimica Organ
results are within 0.4% of the theoretical values.
́
ica “Manuel Lora-Tamayo” (CSIC), and the
1
white solid. mp 230−231 °C. ESI-MS m/z 256 [MH]+. H NMR
General Procedure for the Synthesis of 4-{[Benzyl(methyl)-
amino]methyl}benzonitriles (15−19). A mixture of 4-
(bromomethyl)benzonitrile (5 mmol) and the corresponding amine
(15 mmol) in ethyl ether (50 mL) was refluxed for 10 h. After cooling
to room temperature, a white solid was removed by filtration, and the
solvent was evaporated to dryness under reduced pressure. The
residue was purified on a silica gel column using mixtures of hexane/
EtOAc as eluent, obtaining the corresponding 4-{[benzyl(methyl)-
amino]methyl}benzonitrile as a pure solid. Analytical and spectro-
scopic data of 15 were consistent with those previously described.46
4-{[(2-Chlorobenzyl)(methyl)amino]methyl}benzonitrile
(16). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8
mmol) and 1-(2-chlorophenyl)-N-methylmethanamine (2.23 g, 14.3
mmol). Purification involved the use of hexane/EtOAc (15:1) as
eluent. 16: colorless syrup (1.15 g, 88%). ESI-MS m/z 271 [MH]+,
(CD3OD) δ 8.00 (d, 2H, J = 8.2 Hz), 7.49 (d, 2H, J = 8.2 Hz), 7.39
(m, 5H), 3.98 (s, 2H), 3.95 (s, 2H), 2.43 (s, 3H). 13C NMR (CD3OD)
δ 172.0 (CO), 139.2 (C), 135.7 (C), 135.4 (C), 131.3 (2CH), 131.0
(2CH), 130.9 (2CH), 129.8 (2CH), 129.7 (CH), 61.8 (CH2), 61.3
(CH2), 41.0 (CH3). Purity: 99% (by HPLC). Anal. (C16H18ClNO2) C,
H, N.
N-(4-Carboxybenzyl)-1-(2-chlorophenyl)-N-methylmethana-
minium Chloride (21). From 4-{[(2-chlorobenzyl)(methyl)amino]-
methyl}benzonitrile (16) (1.03 g, 3.8 mmol), 967 mg (88%) of 21 was
obtained as a white solid. mp 144−146 °C. ESI-MS m/z 290 [MH]+,
292 [MH + 2]+. 1H NMR (CD3OD) δ 8.00 (d, 2H, J = 8.4 Hz), 7.57
(dd, 1H, J = 7.0 Hz, J = 2.4 Hz) 7.51 (d, 2H, J = 8.4 Hz), 7.41 (dd, 1H,
J = 7.3 Hz, J = 2.0 Hz), 7.33 (dd, 1H, J = 7.3 Hz, J = 2.0 Hz), 7.30 (dd,
1H, J = 7.3 Hz, J = 2.4 Hz), 3.91 (s, 4H,), 2.38 (s, 3H). 13C NMR
(CD3OD) δ 170.3 (C), 142.2 (C), 135.8 (C), 135.0 (C), 133.0 (CH),
132.8 (CH), 130.9 (2CH), 130.8 (2CH), 130.7 (2CH), 128.3 (CH),
62.2 (CH2), 58.9 (CH2), 41.9 (CH3). Purity: 99% (by HPLC). Anal.
(C16H17Cl2NO2) C, H, N.
N-(4-Carboxybenzyl)-1-(3-chlorophenyl)-N-methylmethana-
minium Chloride (22). From 4-{[(3-chlorobenzyl)(methyl)amino]-
methyl}benzonitrile (17) (1.03 g, 3.8 mmol), 1.07 g (97%) of 22 was
obtained as a white solid. mp 140−142 °C. ESI-MS m/z 290 [MH]+,
292 [MH + 2]+. 1H NMR (CD3OD) δ 8.13 (d, 2H, J = 8.4 Hz), 7.64
(d, 2H, J = 8.4 Hz), 7.61 (s, 1H), 7.51 (m, 3H), 4.48 (s, 2H), 4.42 (s,
2H), 2.73 (s, 3H). 13C NMR (CD3OD) δ 167.5 (C), 135.0 (C), 134.1
(C), 132.6 (C), 131.6 (C), 131.3 (2CH), 131.1 (CH), 130.8 (CH),
130.4 (2CH), 130.3 (CH), 129.6 (CH), 59.3 (CH2), 59.1 (CH2), 38.6
(CH3). Purity: 100% (by HPLC). Anal. (C16H17Cl2NO2) C, H, N.
N-(4-Carboxybenzyl)-1-(2-methoxyphenyl)-N-methylmetha-
naminium Chloride (23). From 4-{[(2-methoxybenzyl)(methyl)-
amino]methyl}benzonitrile (18) (1.01 g, 3.8 mmol), 1.05 g (97%) of
23 was obtained as a white solid. mp 164−166 °C. ESI-MS m/z 286
[MH]+. 1H NMR (CD3OD) δ 8.11 (d, 2H, J = 8.4 Hz), 7.69 (d, 2H, J
= 8.4 Hz), 7.45 (td, 1H, J = 7.7 Hz, J = 1.7 Hz), 7.40 (dd, 1H, J = 7.7
Hz, J = 1.7 Hz), 7.05 (dd, 1H, J = 7.7 Hz, J = 0.9 Hz), 7.00 (td, 1H, J =
7.7, J = 0.9 Hz), 3.80 (s, 3H), 3.29 (s, 2H), 3.23 (s, 2H), 2.81 (s, 3H).
13C NMR (CD3OD) δ 168.8 (C), 159.4 (C), 135.8 (C), 133.6 (C),
133.5 (CH), 132.4 (2CH), 131.4 (2CH), 129.5 (CH), 122.1 (CH),
118.9 (C) 62.2 (CH2), 60.7 (CH3), 56.1 (CH2), 55.9 (CH2) 41.2
(CH3). Purity: 99% (by HPLC). Anal. (C17H20ClNO3) C, H, N.
N-(4-Carboxybenzyl)-1-(3-methoxyphenyl)-N-methylmetha-
naminium Chloride (24). From 4-{[(3-methoxybenzyl)(methyl)-
amino]methyl}benzonitrile (19) (1.01 g, 3.8 mmol), 748 mg (69%) of
24 was obtained as a white solid. mp 100−102 °C. ESI-MS m/z 286
[MH]+. 1H NMR (CD3OD) δ 8.00 (d, 2H, J = 8.3 Hz), 7.47 (d, 2H, J
= 8.3 Hz), 7.31 (t, 1H, J = 7.9 Hz), 7.00 (s, 1H), 6.95 (m, 2H), 3.92 (s,
2H), 3.87 (s, 2H), 3.80 (s, 3H), 2.41 (s, 3H). 13C NMR (CD3OD) δ
161.5 (CO), 139.8 (C), 137.3 (C), 135.4 (C), 130.9 (2CH), 130.8
(2CH), 130.7 (CH), 123.2 (CH), 122.9 (C), 116.5 (CH), 115.0
(CH), 62.0 (CH2), 61.5 (CH2), 55.7 (CH3), 41.3 (CH3). Purity: 99%
(by HPLC). Anal. (C17H20ClNO3) C, H, N.
1
273 [MH + 2]+. H NMR (CDCl3) δ 7.60 (d, 2H, J = 8.4 Hz), 7.51
(dd, 1H, J = 7.6 Hz, J = 1.5 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.36 (dd,
1H, J = 7.6 Hz, J = 1.2 Hz) 7.26 (td, 1H, J = 7.6 Hz, J = 1.2 Hz), 7.20
(td, 1H, J = 7.6 Hz, J = 1.5 Hz), 3.67 (s, 2H), 3.63 (s, 2H), 2.22 (s,
3H). 13C NMR (CDCl3) δ 145.1 (C), 136.3 (C), 134.3 (C), 132.1
(2CH), 130.6 (CH), 129.6 (CH), 129.3 (2CH), 128.3 (2CH), 126.6
(CH), 119.0 (C), 110.7 (C), 61.5 (CH2), 58.7 (CH2), 42.3 (CH3).
Purity: 100% (by HPLC). Anal. (C16H15ClN2) C, H, N.
4-{[(3-Chlorobenzyl)(methyl)amino]methyl}benzonitrile
(17). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8
mmol) and 1-(3-chlorophenyl)-N-methylmethanamine (2.23 g, 14.3
mmol). Purification involved the use of hexane/EtOAc (15:1) as
eluent. 17: colorless syrup (1.29 g, 83%). ESI-MS m/z 271 [MH]+,
273 [MH + 2]+. 1H NMR (CDCl3) δ 7.60 (d, 2H, J = 8.4 Hz), 7.47 (d,
2H, J = 8.4 Hz), 7.36 (s, 1H), 7.26 (m, 3H), 3.55 (s, 2H), 3.50 (s, 2H),
2.17 (s, 3H). 13C NMR (CDCl3) δ 145.2(C), 141.3 (C), 134.5 (C),
132.4 (2CH), 129.9 (CH), 129.5 (2CH), 129.0 (CH), 127.6 (CH),
127.1 (CH), 119.2 (C), 111.1 (C), 61.7 (CH2), 61.6 (CH2), 42.6
(CH3). Purity: 100% (by HPLC). Anal. (C16H15ClN2) C, H, N.
4-{[(2-Methoxybenzyl)(methyl)amino]methyl}benzonitrile
(18). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8
mmol) and 1-(2-methoxyphenyl)-N-methylmethanamine (1.96 g, 14.3
mmol). Purification involved the use of hexane/EtOAc (12:1) as
eluent. 18: colorless syrup (1.58 g, 93%). ESI-MS m/z 267 [MH]+. 1H
NMR (CDCl3) δ 7.60 (d, 2H, J = 8.2 Hz), 7.50 (d, 2H, J = 8.2 Hz),
7.40 (dd, 1H, J = 7.9 Hz, J = 1.6 Hz), 7.25 (td, 1H, J = 7.9 Hz, J = 1.6
Hz), 6.96 (td, 1H, J = 7.9 Hz, J = 0.7 Hz), 6.87 (dd, 1H, J = 7.9 Hz, J =
0.7 Hz), 3.81 (s, 3H), 3.60 (s, 2H), 3.57 (s, 2H), 2.24 (s, 3H). 13C
NMR (CDCl3) δ 158.0 (C), 145.8 (C), 132.3 (2CH), 130.5 (CH),
129.6 (2CH), 128.4 (CH), 126.9 (C), 120.6 (CH), 119.4 (C), 110.8
(C), 110.6 (CH), 61.9 (CH2), 55.6 (CH3), 55.5 (CH2), 42.8 (CH3).
Purity: 100% (by HPLC). Anal. (C17H18N2O) C, H, N.
4-{[(3-Methoxybenzyl)(methyl)amino]methyl}benzonitrile
(19). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8
mmol) and 1-(3-methoxyphenyl)-N-methylmethanamine (1.96 g, 14.3
mmol). Purification involved the use of hexane/EtOAc (12:1) as
eluent. 19: colorless syrup (1.33 g, 77%). ESI-MS m/z 267 [MH]+. 1H
NMR (CDCl3) δ 7.52 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J = 8.4 Hz),
7.18 (t, 1H, J = 8.1 Hz), 6.88 (s, 1H), 6.87 (dd, 1H, J = 8.1 Hz, J = 1.7
Hz), 6.74 (dd, 1H, J = 8.1 Hz, J = 1.7 Hz), 3.74 (s, 3H), 3.47 (s, 2H),
3.45 (s, 2H), 2.12 (s, 3H). 13C NMR (CDCl3) δ 159.5 (C), 145.2 (C),
140.4 (C), 131.9 (2CH), 129.2 (CH), 129.1 (2CH), 120.9 (CH),
118.9 (C), 114.2 (CH), 112.2 (CH), 110.5 (C), 61.8 (CH2), 61.0
(CH2), 55.1 (CH2), 42.3 (CH3). Purity: 100% (by HPLC). Anal.
(C17H18N2O) C, H, N.
General Procedure for the Synthesis of Melatonin− N,N-
Dibenzyl(N-methyl)amino Hybrids (1−14). To a solution of the
corresponding 4-{[benzyl(methyl)amino]methyl}benzoic acid 20−24
(0.5 mmol), triethylamine (1.2 mmol), and PyBOP (0.6 mmol) in
DMF (8 mL) was added the appropriated 2-(1H-indol-3-yl)-
ethanamine (0.5 mmol). The reaction mixture was stirred at room
temperature overnight, and DMF was then evaporated to dryness
under reduced pressure. The residue was dissolved in CH2Cl2 (10 mL)
and consecutively washed with a 10% aqueous NaHCO3 solution (3 ×
10 mL) and H2O (10 mL). The organic phase was dried over sodium
sulfate and evaporated to dryness under reduced pressure. The residue
was purified on a silica gel column using mixtures of hexane/EtOAc as
eluent, obtaining the corresponding dibenzylamino−melatonin de-
rivative as a pure solid.
General Procedure for the Synthesis of 4-{[Benzyl(methyl)-
amino]methyl}benzoic Acids (20−24). To a solution of the
corresponding nitrile (3.8 mmol) in dioxane (6 mL) was added a
solution of NaOH (2 M aq., 29 mL), and the mixture was refluxed for
4 h. After cooling to room temperature, the solution was made acidic
with HCl (2 M aqueous), and the resulting acid was isolated by
filtration.
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N-[2-(1H-Indol-3-yl)ethyl]-4-{[benzyl(methyl)amino]methyl}-
benzamide (1). Reagents used were 4-{[benzyl(methyl)amino]-
methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2-(1H-indol-3-
yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol),
and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/
EtOAc (1:3) as eluent. Hybrid 1: white solid. mp 94−96 °C (91.4 mg,
4-{[Benzyl(methyl)amino]methyl}-N-[2-(6-fluoro-1H-indol-3-
yl)ethyl]benzamide (5). Reagents used were 4-{[benzyl(methyl)-
amino]methyl}benzoic acid (20) (127.63 mg, 0.5 mmol), 2-(6-fluoro-
1H-indol-3-yl)ethanamine (89.0 mg, 0.5 mmol), PyBOP (312.2 mg,
0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the
use of hexane/EtOAc (2:3) as eluent. Hybrid 5: yellow solid. mp 83−
85 °C (135 mg, 65%). ESI-MS m/z 416 [MH]+. 1H NMR (CDCl3) δ
8.63 (s, NH), 7.63 (d, 2H, J = 8.2 Hz), 7.49 (dd, 1H, J = 9.0 Hz, J =
5.2 Hz), 7.36 (d, 2H, J = 8.2 Hz), 7.32 (m, 4H), 7.24 (m, 1H), 6.99
(dd, 1H, J = 9.7 Hz, J = 2.3 Hz), 6.96 (d, 1H, J = 2.3 Hz), 6.84 (td, 1H,
J = 9.0 Hz, J = 2.3 Hz), 6.40 (t, 1H, J = 5.7 Hz), 3.74 (q, 2H, J = 6.7
Hz), 3.24 (s, 2H), 3.51 (s, 2H), 3.02 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H).
13C NMR (CDCl3) δ 167.5 (CONH), 161.1 (C), 158.8 (C), 143.0
(C), 138.7 (C), 136.3 (C, J=12.2), 128.9 (2CH), 128.8 (2CH), 128.3
(2CH), 127.1 (CH), 126.8 (2CH), 123.9 (C), 122.4 (CH), 122.3
(CH), 119.4 (C), 119.3 (CH), 112.8 (CH), 108.0 (CH, JC,F = 24.4
Hz), 97.6 (CH, JC,F = 25.9 Hz), 61.8 (CH2), 61.2 (CH2), 42.1 (CH3),
40.2 (CH3), 25.2 (CH2). Purity: 98% (by HPLC). Anal.
(C26H26FN3O) C, H, N.
N-[2-(1H-Indol-3-yl)ethyl]-4-{[(2-chlorobenzyl)(methyl)-
amino]methyl} benzamide (6). Reagents used were 4-{[(2-
chlorobenzyl)(methyl)amino]methyl}benzoic acid (21) (135.4 mg,
0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol),
PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (1:1) as eluent.
Compound 6: white solid. mp 97−99 °C (106 mg, 49%). ESI-MS m/z
432 [MH]+, 434 [MH + 2]+. 1H NMR (CDCl3) δ 8.20 (s, NH), 7.65
(d, 1H, J = 8.1 Hz), 7.62 (d, 2H, J = 8.3 Hz), 7.52 (dd, 1H, J = 7.6 Hz,
J = 1.2 Hz), 7.38 (d, 2H, J = 8.3 Hz), 7.37 (d, 1H, J = 8.1 Hz), 7.34
(dd, 1H, J = 7.8 Hz, J = 1.0 Hz).7.21 (m, 4H), 7.13 (t, 1H, J = 7.2 Hz),
7.06 (d, J = 2.0 Hz), 6.23 (t, NH, J = 5.4 Hz), 3.80 (q, 2H, J = 6.6 Hz),
3.63 (s, 2H), 3.60 (s, 2H), 3.09 (t, 2H, J = 6.6 Hz), 2.19 (s, 3H). 13C
NMR (CDCl3) δ 167.3 (CONH), 143.0 (C), 136.5 (C), 136.4 (C),
134.2 (C), 133.4 (C), 130.6 (CH), 129.4 (CH), 128.9 (2CH), 128.1
(CH), 127.2 (CH), 126.8 (2CH), 126.6 (CH), 122.2 (CH), 122.1
(CH), 119.5 (CH), 118.7 (CH), 113.0 (C), 111.3 (CH), 61.7 (CH2),
58.5 (CH2), 42.2 (CH3), 40.2 (CH2), 25.3 (CH2). Purity: 100% (by
HPLC). Anal. (C26H26ClN3O) C, H, N.
1
46%). ESI-MS m/z 398 [MH]+. H NMR (CDCl3) δ 8.20 (s, NH),
7.65 (s, 1H), 7.62 (d, 2H, J = 8.2 Hz), 7.37 (d, 2H, J = 8.2 Hz), 7.33
(m, 5H), 7.25 (m, 1H), 7.21 (td, 1H, J = 7.1 Hz, J = 1.0 Hz), 7.12 (td,
1H, J = 7.9 Hz, J = 1.0 Hz), 7.05 (m, 1H), 6.22 (t, NH, J = 5.4 Hz),
3.79 (q, 2H, J = 6.6 Hz), 3.53 (s, 2H), 3.51 (s, 2H), 3.09 (t, 2H, J = 6.6
Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 142.9 (C),
138.8 (C), 136.4 (C), 133.4 (C), 128.9 (2CH), 128.9 (2CH), 128.3
(2CH), 127.3 (C), 127.1 (CH), 126.8 (2CH), 122.2 (CH), 122.1
(CH), 119.5 (CH), 118.7 (CH), 113.0 (C), 111.3 (CH), 61.8 (CH2),
61.3 (CH2), 42.2 (CH3), 40.2 (CH2), 25.3 (CH2). Purity: 99% (by
HPLC). Anal. (C26H27N3O) C, H, N.
4-{[Benzyl(methyl)amino]methyl}-N-[2-(5-hydroxy-1H-indol-
3-yl)ethyl]benzamide (2). Reagents used were 4-{[benzyl(methyl)-
amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 3-(2-
aminoethyl)-1H-indol-5-ol (88.0 mg, 0.5 mmol), PyBOP (312.2 mg,
0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the
use of hexane/EtOAc (2:1) as eluent. Compound 2: white solid. mp
75−77 °C (62.0 mg, 30%). ESI-MS m/z 414 [MH]+. 1H NMR
(CDCl3) δ 7.74 (d, 2H, J = 8.2 Hz), 7.41 (d, 2H, J = 8.2 Hz), 7.32 (m,
4H), 7.24 (t, 1H, J = 7.1 Hz), 7.15 (d, 1H, J = 8.5 Hz), 7.03 (s, 1H),
6.99 (d, 1H, J = 2.2 Hz), 6.66 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz), 3.63 (d,
2H, J = 7.4 Hz), 3.54 (s, 2H), 3.51 (s, 2H), 2.98 (t, 2H, J = 7.4 Hz),
2.16 (s, 3H). 13C NMR (CDCl3) δ 170.6 (CONH), 151.7 (C), 144.2
(C), 140.1 (C), 135.3 (C), 133.6 (C), 130.8 (2CH), 130.7 (2CH),
130.0 (C), 129.8 (2CH), 128.8 (CH), 128.7 (2CH), 124.7 (CH),
113.2 (CH), 113.1 (C), 112.9 (CH), 104.1 (CH), 63.3 (CH2), 62.7
(CH2), 42.9 (CH3), 42.5 (CH2), 26.8 (CH2). Purity: 99% (by HPLC).
Anal. (C26H27N3O2) C, H, N.
4-{[Benzyl(methyl)amino]methyl}-N-[2-(5-methoxy-1H-
indol-3-yl)ethyl]benzamide (3). Reagents used were 4-{[benzyl-
(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2-
(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP
(312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification
involved the use of hexane/EtOAc (1:1) as eluent. Hybrid 3: yellow
4-{[(2-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(5-me-
thoxy-1H-indol-3-yl)ethyl]benzamide (7). Reagents used were 4-
{[(2-chlorobenzyl)(methyl)amino]methyl}benzoic acid (21) (135.4
mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5
mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (1:1) as eluent. Hybrid
7: white solid. mp 120−122 °C (132 mg, 57%). ESI-MS m/z 462
1
solid. mp 102−103 °C (154 mg, 72%). ESI-MS m/z 428 [MH]+. H
NMR (CDCl3) δ 8.68 (s, NH), 7.66 (d, 2H, J = 8.2 Hz), 7.36 (d, 2H, J
= 8.2 Hz), 7.32 (m, 4H), 7.20 (d, 1H, J = 8.8 Hz), 7.04 (d, 1H, J = 2.3
Hz), 6.95 (d, 1H, J = 2.3 Hz), 6.84 (dd, 1H, J = 8.8 Hz, J = 2.3 Hz),
6.52 (t, NH, J = 5.5 Hz), 3.75 (q, 2H, J = 6.7 Hz), 3.74 (s, 3H), 3.51
(s, 4H), 3.03 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ
167.4 (CONH), 153.8 (C), 143.0 (C), 138.8 (C), 133.1 (C), 131.5
(C), 128.8 (2CH), 128.8 (2CH), 128.2 (2CH), 127.6 (C), 127.0
(CH), 126.8 (2CH), 123.0 (CH), 112.3 (C), 112.2 (CH), 112.1
(CH), 100.2 (CH), 61.7 (CH2), 61.1 (CH2), 55.6 (CH3), 42.1 (CH3),
40.4 (CH2), 25.2 (CH2). Purity: 98% (by HPLC). Anal.
(C27H29N3O2) C, H, N.
1
[MH]+, 464 [MH + 2]+. H NMR (CD3OD) δ 7.73 (d, 2H, J = 8.3
Hz), 7.54 (d, 1H, J = 8.3 Hz), 7.46 (d, 1H, J = 8.3 Hz), 7.44 (d, 2H, J
= 8.6 Hz), 7.32 (d, 1H, J = 8.3 Hz), 7.36 (m, 1H), 7.21 (d, 1H, J = 8.8
Hz), 6.72 (dd, 1H, J = 8.8 Hz, J = 2.4 Hz), 3.72 (s, 3H), 3.66 (s, 2H),
3.63 (s, 2H), 3.34 (t, 2H, J = 7.3 Hz), 3.03 (t, 2H, J = 7.3 Hz), 2.20 (s,
3H). 13C NMR (CD3OD) δ 170.1 (CONH), 154.9 (C), 137.5 (C),
135.5 (C), 134.8 (C), 133.3 (C), 132.3 (C), 132.3 (CH), 130.5 (CH),
130.4 (CH), 130.3 (C), 130.2 (2CH), 129.7 (CH), 129.2 (C), 128.3
(2CH), 113.3 (C), 112.9 (CH), 112.7 (CH), 101.2 (CH), 62.6 (CH2),
59.4 (CH2), 56.1 (CH3), 42.5 (CH2), 42.3 (CH3), 26.3 (CH2). Purity:
99% (by HPLC). Anal. (C27H28ClN3O2) C, H, N.
N-[2-(1H-Indol-3-yl)ethyl]-4-{[(3-chlorobenzyl)(methyl)-
amino]methyl}benzamide (8). Reagents used were 4-{[(3-
chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4 mg,
0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol),
PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (2:1) as eluent.
Derivative 8: white solid. mp 110−112 °C (119 mg, 55%). ESI-MS m/
z 432 [MH]+, 434 [MH + 2]+. 1H NMR (CDCl3) δ 8.21(s, NH), 7.64
(d, 2H, J = 8.3 Hz), 7.64 (m, 1H), 7.37 (d, 2H, J = 8.3 Hz), 7.37 (m,
2H), 7.23 (m, 4H), 7.13 (td, 1H, J = 7.5 Hz, J = 1.0 Hz), 7.06 (d, 1H, J
= 2.5 Hz), 6.23 (t, NH, J = 5.5 Hz), 3.80 (q, 2H, J = 6.7 Hz), 3.52 (s,
2H), 3.46 (s, 2H), 3.09 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H). 13C NMR
(CDCl3) δ 167.4 (CONH), 142.8 (C), 141.3 (C), 136.4 (C), 134.2
(C), 133.5 (C), 129.5 (CH), 128.8 (2CH), 128.7 (2CH), 127.3 (C),
4-{[Benzyl(methyl)amino]methyl}-N-[2-(6-methoxy-1H-
indol-3-yl)ethyl]benzamide (4). Reagents used were 4-{[benzyl-
(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2-
(6-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP
(312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification
involved the use of hexane/EtOAc (1:1) as eluent. Derivative 4: yellow
1
solid. mp 119−121 °C (139 mg, 65%). ESI-MS m/z 428 [MH]+. H
NMR (acetone-d6) δ 7.87 (d, 2H, J = 8.2 Hz), 7.49 (d, 1H, J = 8.6
Hz), 7.44 (d, 2H, J = 8.2 Hz), 7.37 (m, 2H), 7.31 (m, 2H), 7.23 (m,
1H), 7.05 (s, 1H), 6.90 (d, 1H, J = 2.2 Hz), 6.68 (dd, 1H, J = 8.6 Hz, J
= 2.2 Hz), 3.76 (s, 3H), 3.69 (t, 2H, J = 7.4 Hz), 3.54 (s, 2H), 3.51 (s,
2H), 3.02 (t, 2H, J = 7.4 Hz), 2.12 (s, 3H). 13C NMR (acetone-d6) δ
167.2 (CONH), 157.2 (C), 143.7 (C), 140.2 (C), 138.2 (C), 134.7
(C), 129.5 (2CH), 129.3 (2CH), 129.0 (2CH), 127.9 (2CH), 127.7
(CH), 122.9 (C), 121.7 (CH), 119.9 (CH), 113.4 (C), 109.6 (CH),
95.2 (CH), 62.3 (CH2), 61.9 (CH2), 55.6 (CH3), 42.3 (CH3), 41.2
(CH2), 26.3 (CH2). Purity: 99% (by HPLC). Anal. (C27H29N3O2) C,
H, N.
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127.2 (CH), 126.9 (2CH), 122.2 (CH), 122.1 (CH), 119.5 (CH),
118.7 (CH), 113.0 (C), 111.3 (CH), 61.4 (CH2), 61.2 (CH2), 42.3
(CH3), 40.2 (CH2), 25.3 (CH2). Purity: 100% (by HPLC). Anal.
(C26H26ClN3O) C, H, N.
J = 7.7 Hz, J = 1.0 Hz), 6.85 (m, 2H), 6.27 (t, NH, J = 5.6 Hz), 3.79 (s,
3H), 3.77 (s, 3H), 3.75 (q, 2H, J = 6.6 Hz), 3.58 (s, 2H) 3.55 (s, 2H),
3.05 (t, 2H, J = 6.6 Hz), 2.20 (s, 3H). 13C NMR (CDCl3) δ 167.4
(CONH), 157.7 (C), 154.0 (C), 143.3 (C), 133.2 (C), 131.5 (C),
130.3 (CH), 128.9 (2CH), 128.0 (CH), 127.7 (C), 126.8 (C), 126.7
(2CH), 122.9 (CH), 120.3 (CH), 112.8 (C), 112.5 (CH), 112.0
(CH), 110.3 (CH), 100.3 (CH), 61.7 (CH2), 55.8 (CH2), 55.3 (CH3),
55.2 (CH3), 42.4 (CH3), 40.3 (CH2), 25.3 (CH2). Purity: 100% (by
HPLC). Anal. (C28H31N3O3) C, H, N.
N-[2-(1H-Indol-3-yl)ethyl]-4-{[(3-methoxybenzyl)(methyl)-
amino]methyl}benzamide (13). Reagents used were 4-{[(3-
methoxybenzyl)(methyl)amino]methyl}benzoic acid (24) (142.7 mg,
0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol),
PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of EtOAc as eluent. Compound 13:
yellow solid. mp 81−83 °C (166 mg, 78%). ESI-MS m/z 428 [MH]+.
1H NMR (CD3OD) δ 7.74 (d, 2H, J = 8.4 Hz), 7.60 (dd, 1H, J = 8.1
Hz, J = 1.0 Hz), 7.42 (d, 2H, J = 8.4 Hz), 7.32 (dd, 1H, J = 8.1 Hz, J =
1.0 Hz), 7.22 (t, 1H, J = 8.1 Hz), 7.09 (s, IH), 7.06 (td, 1H, J = 8.1 Hz,
J = 1.0 Hz), 6.98 (td, 1H, J = 8.1 Hz, J = 1.0 Hz), 6.93 (d, 1H, J = 2.0
Hz), 6.91 (d, 1H, J = 8.1 Hz), 6.80 (dd, 1H, J = 8.1 Hz, J = 2.0 Hz),
3.77 (s, 3H), 3.66 (t, 2H, J = 7.4 Hz), 3.53 (s, 2H) 3.48 (s, 2H), 3.05
(t, 2H, J = 7.4 Hz), 2.16 (s, 3H). 13C NMR (CD3OD) δ 170.1
(CONH), 161.2 (C), 143.8 (C), 141.3 (C), 138.1 (C), 134.8 (C),
130.3 (CH), 130.3 (2CH), 128.8 (C), 128.3 (2CH), 123.4 (CH),
122.5 (CH), 122.3 (CH), 119.6 (CH), 119.4 (CH), 115.7 (CH),
113.7 (CH), 113.4 (C), 112.2(CH), 62.7 (CH2), 62.1 (CH2), 55.6
(CH3), 42.5 (CH3), 42.2 (CH2), 26.3 (CH2). Purity: 99% (by HPLC).
Anal. (C27H29N3O2) C, H, N.
4-{[(3-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(5-me-
thoxy-1H-indol-3-yl)ethyl]benzamide (9). Reagents used were 4-
{[(3-chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4
mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5
mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (2:1) as eluent. Hybrid
9: yellow solid. mp 108−110 °C (119 mg, 55%). ESI-MS m/z 462
1
[MH]+, 464 [MH + 2]+. H NMR (CDCl3) δ 8.15 (s, NH), 7.62 (d,
2H, J = 8.3 Hz), 7.35 (d, 2H, J = 8.3 Hz), 7.34 (s, 2H), 7.25 (d, 1H, J =
8.6 Hz), 7.22 (m, 3H), 7.03 (s. 2H), 6.85 (dd, 1H, J = 8.6 Hz, J = 2.3
Hz), 6.27 (t, NH, J = 5.6 Hz), 3.77 (q, 2H, J = 6.5 Hz), 3.86 (s, 3H),
3.51 (s, 2H) 3.45 (s, 2H), 3.04 (t, 2H, J = 6.5 Hz), 2.14 (s, 3H). 13C
NMR (CDCl3) δ 167.6 (CONH), 154.3 (C), 143.1 (C), 141.5 (C),
134.4 (C), 133.6 (C), 131.7 (C), 129.8 (CH), 129.1 (2CH), 129.0
(2CH), 127.9 (C), 127.5 (CH), 127.2 (2CH), 123.2 (CH), 113.0 (C),
112.8 (CH), 112.3 (CH), 100.5 (CH), 61.7 (CH2), 61.4 (CH2), 56.0
(CH3), 42.5 (CH3), 40.5 (CH2), 25.5 (CH2). Purity: 100% (by
HPLC). Anal. (C27H28ClN3O2) C, H, N.
4-{[(3-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(6-me-
thoxy-1H-indol-3-yl)ethyl]benzamide (10). Reagents used were 4-
{[(3-chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4
mg, 0.5 mmol), 2-(6-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5
mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (1:1) as eluent. Hybrid
10: yellow solid. mp 58−60 °C (189 mg, 82%). ESI-MS m/z 462
1
[MH]+, 464 [MH + 2]+. H NMR (CDCl3) δ 8.31 (s, NH), 7.65 (d,
N - [ 2 - ( 5 - M e t h o x y - 1 H - i n d o l - 3 - y l ) e t h y l ] - 4 - { [ ( 3 -
methoxybenzyl)(methyl)amino]methyl}benzamide (14). Re-
agents used were 4-{[(3-methoxybenzyl)(methyl)amino]methyl}-
benzoic acid (24) (142.7 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-
yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol),
and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/
EtOAc (2:1) as eluent. Hybrid 14: white solid. mp 82−84 °C (144 mg,
2H, J = 8.4 Hz), 7.48 (d, 1H, J = 8.6 Hz), 7.36 (d, 2H, J = 8.4 Hz),
7.35 (s, 1H), 7.23 (m, 3H), 6.91 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J =
2.2 Hz), 6.78 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz), 6.35 (t, NH, J = 5.6
Hz), 3.81 (s, 3H), 3.75 (q, 2H, J = 6.6 Hz), 3.52 (s, 2H) 3.46 (s, 2H),
3.03 (t, 2H, J = 6.6 Hz), 2.15 (s, 3H). 13C NMR (CDCl3) δ 167.4
(CONH), 156.5 (C), 142.7 (C), 141.2 (C), 137.2 (C), 134.1 (C),
133.4 (C), 129.85 (CH), 128.8 (2CH), 128.7 (2CH), 127.2 (CH),
126.9 (2CH), 121.6 (C), 120.9 (CH), 119.3 (C), 112.7 (C), 109.4
(CH), 94.7 (CH), 61.4 (CH2), 61.2 (CH2), 55.6 (CH3), 42.2 (CH3),
40.2 (CH2), 25.3 (CH2). Purity: 100% (by HPLC). Anal.
(C27H28ClN3O2) C, H, N.
N-[2-(1H-Indol-3-yl)ethyl]-4-{[(2-methoxybenzyl)(methyl)-
amino]methyl}benzamide (11). Reagents used were 4-{[(2-
methoxybenzyl)(methyl)amino]methyl}benzoic acid (23) (142.7 mg,
0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol),
PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol).
Purification involved the use of hexane/EtOAc (1:1) as eluent.
Compound 11: white solid. mp 80−82 °C (130 mg, 61%). ESI-MS m/
z 428 [MH]+. 1H NMR (CD3OD) δ 7.74 (d, 2H, J = 8.3 Hz), 7.60 (d,
1H, J = 8.1 Hz), 7.42 (d, 2H, J = 8.3 Hz), 7.31 (m, 2H), 7.24 (td, 1H, J
= 7.8 Hz, J = 1.7 Hz), 7.09 (s, 1H), 7.06 (m, 1H), 6.97 (td, 1H, J = 8.1
Hz, J = 1.0 Hz), 6.93 (m, 1H), 6.91 (td, 1H, J = 7.6 Hz, J = 1.0 Hz),
3.78 (s, 3H), 3.66 (t, 2H, J = 7.4 Hz), 3.54 (s, 2H) 3.34 (s, 2H), 3.06
(t, 2H, J = 7.4 Hz), 2.18 (s, 3H). 13C NMR (CD3OD) δ 170.1
(CONH), 159.4 (C), 143.7 (C), 138.1 (C), 134.7 (C), 132.1 (CH),
130.4 (2CH), 129.7 (CH), 128.8 (C), 128.2 (2CH), 127.0 (C), 123.4
(CH), 122.3 (CH), 121.2 (CH), 119.6 (CH), 119.4 (CH), 113.4 (C),
112.2 (CH), 111.6 (CH), 62.5 (CH2), 56.0 (CH3), 55.7 (CH2), 42.6
(CH3), 42.2 (CH2), 26.3 (CH2). Purity: 100% (by HPLC). Anal.
(C27H29N3O3) C, H, N.
1
63%). ESI-MS m/z 458 [MH]+. H NMR (CD3OD) δ 7.71 (d, 2H, J
= 8.3 Hz), 7.38 (d, 2H, J = 8.3 Hz), 7.21 (d, 1H, J = 8.7 Hz), 7.20 (t,
1H, J = 7.6 Hz), 7.06 (d, 1H, J = 2.4 Hz), 7.05 (s, 1H), 6.90 (d, 1H, J =
1.2 Hz), 6.88 (d, 1H, J = 7.6 Hz), 6.78 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz),
6.72 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz), 3.75 (s, 3H), 3.70 (s, 3H), 3.63
(t, 2H, J = 7.3 Hz), 3.49 (s, 2H) 3.44 (s, 2H), 3.02 (t, 2H, J = 7.3 Hz),
2.13 (s, 3H). 13C NMR (CD3OD) δ 170.0 (CONH), 161.2 (C), 154.9
(C), 143.8 (C), 141.3 (C), 134.7 (C), 133.3 (C), 130.3 (CH), 130.2
(2CH), 129.2 (C), 128.3 (2CH), 124.2 (CH), 122.5 (CH), 115.6
(CH), 113.7 (CH), 113.3 (C), 112.9 (CH), 112.7 (CH), 101.2 (CH),
62.7 (CH2), 62.1 (CH2), 56.1 (CH3), 55.6 (CH3), 42.5 (CH2), 42.3
(CH3), 26.3 (CH2). Purity: 100% (by HPLC). Anal. (C28H31N3O3) C,
H, N.
Biochemical Studies. Inhibition of Human AChE and BuChE.
The method of Ellman et al. was followed.47 The assay solution
consisted of 0.1 M phosphate buffer, pH 8.0, 400 μM 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB, Ellman’s reagent), 0.05 U mL−1 h-AChE
(human recombinant acetylcholinesterase, Sigma Chemical Co.) or
0.024 U mL−1 h-BuChE (butyrylcholinesterase from human serum,
Sigma Chemical Co.), and 800 μM acetylthiocholine iodide or 500 μM
butyrylthiocholine as the substrate of the enzymatic reaction,
respectively. The compounds tested were added to the assay solution
and preincubated with the enzyme for 5 min at 30 °C. After that
period, the substrate was added. The absorbance changes at 412 nm
were recorded for 5 min with a UV/vis microplate spectrophotometer
(Multiskan Spectrum, Thermo Electron Co.). The reaction rates were
compared, and the inhibition percentage resulting from the presence
of test compound was calculated. The IC50 is defined as the
concentration of each compound that reduces 50% of the enzymatic
activity without any inhibitor.
N - [ 2 - ( 5 - M e t h o x y - 1 H - i n d o l - 3 - y l ) e t h y l ] - 4 - { [ ( 2 -
methoxybenzyl)(methyl)amino]methyl}benzamide (12). Re-
agents used were 4-{[(2-methoxybenzyl)(methyl)amino]methyl}-
benzoic acid (23) (142.7 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-
yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol),
and Et3N (156 μL, 1.2 mmol). Purification involved the use of EtOAc
as eluent. Hybrid 12: yellow solid. mp 58−60 °C (174 mg, 76%). ESI-
MS m/z 458 [MH]+. 1H NMR (CDCl3) δ 8.18 (s, NH), 7.62 (d, 2H, J
= 8.6 Hz), 7.40 (m, 1H), 7.39 (d, 2H, J = 8.6 Hz), 7.25 (d, 1H, J = 9.0
Hz), 7.22 (td, 1H, J = 7.7 Hz, J = 2.0 Hz), 7.03 (m, 2H), 6.94 (td, 1H,
Measurement of Propidium Iodide Displacement from the
Peripheral Anionic Site (PAS) of AChE. A soltion 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 were added to obtain final
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concentrations of 0.3, 1.0, and 3.0 μM, and the solutions were kept at
room temperature for at least 6 h. Afterward, 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.
penicillin, and 50 μg mL−1 streptomycin (reagents from GIBCO,
Madrid, Spain). Cultures were seeded into flasks containing
supplemented medium and were maintained at 37 °C in 5% CO2/
humidified air. Stock cultures were passaged 1:4 twice weekly. For
assays, SH-SY5Y cells were subcultured in 48-well plates at a seeding
density of 105 cells per well. For the cytotoxicity experiments, cells
were treated with drugs before they reached confluence in DMEM free
of serum.
Cell Viability Experiments. To study the cytotoxic effects of
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 1 μM, and the quantitative assessment of cell
death was made by measurement of the percent of the intracellular
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).
Neuroprotection Studies. To study the cytoprotective action of
the compounds against cell death induced by the mixture of rotenone
(30 μM) and oligomycin A (10 μM), drugs were given at time zero
and maintained for 24 h. Then, the media were replaced by fresh
media that still contained the drug plus the cytotoxic insult, which was
left for an additional 24 h period. Thereafter, cell survival was assessed
by measuring LDH activity.
Measurement of Lactic Dehydrogenase (LDH) Activity.
Extracellular and intracellular LDH activity was measured by UV/vis
using a cytotoxicity cell death kit (Roche-Boehringer Mannheim,
Germany) according to the manufacturer’s indications. 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 relative to 100% of the extracellular LDH released by
the vehicle with respect to the total. To determine 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
normalized to 100%, and that value was subtracted from 100.
Measurement of Cell Viability by MTT Assay. Cell viability was
also measured by quantitative colorimetric assay with 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)
(Sigma-Aldrich), as described previously.60 Briefly, 50 μL of the
MTT labeling reagent, at a final concentration of 0.5 mg mL−1, was
added to each well at the end of the incubation period, and the plate
was placed in a humidified incubator at 37 °C with 5% CO2 and 95%
air (v/v) for an additional 2 h. Then, the insoluble formazan was
dissolved with dimethyl sulfoxide; colorimetric determination of MTT
reduction was measured at 540 nm. Control cells treated with 0.1%
DMSO were taken as 100% viability.
In Vitro Blood−Brain Barrier Permeation Assay (PAMPA-
BBB). Prediction of the brain penetration was evaluated using a
parallel artificial membrane permeation assay (PAMPA-BBB) in a
similar manner as previously described.38,41,51 Pipetting was performed
with a semi-automatic robot (CyBi-SELMA), and UV absorbance was
read with a microplate spectrophotometer (Multiskan Spectrum,
Thermo Electron Co.). 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 200 μL of PBS/ethanol (70:30), and the filter surface of the
donor microplate was impregnated with 4 μL of PBL in dodecane (20
mg mL−1). Compounds were dissolved in PBS/ethanol (70:30) at 100
μg mL−1, filtered through a Millex filter, and then added to the donor
wells (200 μL). The donor filter plate was carefully placed on the
acceptor plate to form a sandwich, which was left undisturbed for 240
min at 25 °C. After incubation, the donor plate was carefully removed,
and the concentration of compounds in the acceptor wells was
determined by UV/vis spectroscopy. Every sample was analyzed at five
wavelengths in four wells and in at least three independent runs, and
the results are given as the mean
standard deviation. In each
experiment, 11 quality control standards of known BBB permeability
were included to validate the analysis set.
Oxygen Radical Absorbance Capacity Assay. The ORAC
method was followed52,59 using a Polarstar Galaxy plate reader (BMG
Labtechnologies GmbH, Offenburg, Germany) with 485-P excitation
and 520-P emission filters. The equipment was controlled by
Fluorostar Galaxy software (version 4.11-0) for fluorescence measure-
ment. 2,2′-Azobis(amidinopropane) dihydrochloride (AAPH), ( )-6-
hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), and
fluorescein (FL) were purchased from Sigma-Aldrich. The reaction
was carried out in 75 mM phosphate buffer (pH 7.4), and the final
reaction mixture was 200 μL. Antioxidant (20 μL) and FL (120 μL; 70
mM, final concentration) solutions were placed in a black 96-well
microplate (96F untreat, Nunc). The mixture was preincubated for 15
min at 37 °C, and AAPH solution (60 μL, 12 mM, final concentration)
was then added rapidly using a multichannel pipet. The microplate was
immediately placed in the reader, and the fluorescence was recorded
every minute for 80 min. The microplate was automatically shaken
prior each reading. Samples were measured at eight different
concentrations (0.1−1 μM). A blank (FL + AAPH in phosphate
buffer), instead of the sample solution, and eight calibration solutions
using trolox (1−8 μM) were also carried out in each assay. All reaction
mixtures were prepared in duplicate, and at least three independent
assays were performed for each sample. Raw data were exported from
Fluostar Galaxy Software to an Excel sheet for further calculations.
Antioxidant curves (fluorescence vs time) were first normalized to the
curve of the blank corresponding to the same assay, and the area under
the fluorescence decay curve (AUC) was calculated. The net AUC
corresponding to a sample was calculated by subtracting the AUC
corresponding to the blank. Regression equations between net AUC
and antioxidant concentration were calculated for all samples. ORAC-
FL values were expressed as trolox equivalents by using the standard
curve calculated for each assay, where the ORAC-FL value of trolox
was taken to be 1.0.
Neurogenesis Assays. Animals. Adult (8−12 weeks old) male
Wistar rats (n = 6 per group), housed in a 12 h light−dark cycle animal
facility, were used in this study. All procedures with animals were
specifically approved by the Ethics Committee for Animal Exper-
imentation of the CSIC and carried out in accordance with National
(normative 1201/2005) and International recommendations (Direc-
tive 2010/63 from the European Communities Council). Special care
was taken to minimize animal suffering.
Neurosphere Cultures. Neurospheres (NS) were derived from
the subgranular zone of the dentate gyrus of the hippocampus of adult
Wistar rats, which were induced to proliferate using established
passaging methods to achieve optimal cellular expansion according to
published protocols.56 Rats were decapitated, and brains were
dissected, obtaining the hippocampus as described.57 Briefly, cells
were seeded into 12-well dishes and cultured in DMEM/F12 (1:1,
Invitrogen) containing 10 ng mL−1 epidermal growth factor (EGF,
Peprotech, London, UK), 10 ng mL−1 fibroblast growth factor (FGF,
Peprotech), and B27 medium (Gibco). After 3 days in culture, primary
NS cultures were treated with different compounds. To determine the
ability of compounds to induce differentiation, NS from 10 day old
Studies of Cell Viability and Neuroprotection. Culture of SH-
SY5Y Cells. SH-SY5Y cells, at passages between 3 and 16 after
defreezing, were maintained in a Dulbecco’s modified Eagle’s medium
(DMEM) containing 15 nonessential amino acids and supplemented
with 10% fetal calf serum (FCS), 1 mM glutamine, 50 units mL−1
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Article
cultures were plated onto 100 μg mL−1 poly-L-lysine-coated coverslips
and treated for 48 h in the presence of serum but in the absence of
exogenous growth factors.
ligand; ORAC-FL, oxygen radical absorbance capacity by
fluorescence; PAMPA-BBB, parallel artificial membrane per-
meation assay for blood−brain barrier permeation; PAS,
peripheral anionic site; PyBOP, benzotriazol-1-yl-oxytripyrroli-
dinophosphonium hexafluorophosphate
Immunocytochemistry. After treatment, cells were processed for
immunocytochemistry with two types of neurogenesis-associated
neuronal markers: anti-β-tubulin antibody (clone TuJ1), associated
with early stages of neurogenesis, and MAP-2 (microtubule-associated
protein 2), a marker of neuronal maturation. DAPI staining (4′,6-
diamidino-2-phenylindole) was used as a nuclear marker. Basal values
were obtained under the same conditions but in the absence of any
product. Melatonin (endogenous ligand of the melatoninergic
receptors) and luzindole (melatoninergic antagonist) were used as
controls. The images were obtained using a Nikon fluorescence
microscope 90i that was coupled to a digital camera Qi. The
microscope configuration was adjusted to produce the optimum
signal-to-noise ratio. For quantifying the number of cells producing a
given marker in any experiment, the number of positive cells in the
neurosphere was counted as previously described.58 Cell numbers
were estimated from a total of five neurospheres per condition over
three independent experiments.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analyses of new melatonin−N,N-dibenzyl(N-
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Corresponding Author
amyloid aggregation and acetylcholinesterase as multi-target anti-
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■
*Phone: 34-91-5622900. Fax: 34-91-5644853. E-mail:
isabelrguez@iqm.csic.es.
́
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We gratefully acknowledge the financial support of the Spanish
Ministry of Economy and Competitiveness (projects SAF2012-
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Aβ, β-amyloid peptide; ACh, acetylcholine; AChE, acetylcho-
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BBB, blood−brain barrier; BuChE, butyrylcholinesterase; CAS,
catalytic active site; CNS, central nervous system; hAChE,
human AChE; hBuChE, human BuChE; MTL, multitarget
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