Synthesis, biological evaluation, and molecular modeling of donepezil and N-[(5-(Benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine hybrids as new multipotent cholinesterase/monoamine oxidase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1021/jm200853t

A new family of multitarget molecules able to interact with acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as with monoamino oxidase (MAO) A and B, has been synthesized. Novel compounds (3-9) have been designed using a conjunctive approach that combines the benzylpiperidine moiety of the AChE inhibitor donepezil (1) and the indolyl propargylamino moiety of the MAO inhibitor N-[(5-benzyloxy-1-methyl-1H-indol-2- yl)methyl]-N-methylprop-2-yn-1-amine (2), connected through an oligomethylene linker. The most promising hybrid (5) is a potent inhibitor of both MAO-A (IC₅₀ = 5.2 ± 1.1 nM) and MAO-B (IC₅₀ = 43 ± 8.0 nM) and is a moderately potent inhibitor of AChE (IC₅₀ = 0.35 ± 0.01 μM) and BuChE (IC₅₀ = 0.46 ± 0.06 μM). Moreover, molecular modeling and kinetic studies support the dual binding site to AChE, which explains the inhibitory effect exerted on Aβ aggregation. Overall, the results suggest that the new compounds are promising multitarget drug candidates with potential impact for Alzheimer's disease therapy. (Figure presented)

Full text of DOI:10.1021/jm200853t

Article
pubs.acs.org/jmc
Synthesis, Biological Evaluation, and Molecular Modeling of
Donepezil and N-[(5-(Benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-
methylprop-2-yn-1-amine Hybrids as New Multipotent
Cholinesterase/Monoamine Oxidase Inhibitors for the Treatment of
Alzheimer’s Disease
∥
§
‡
Irene Bolea,†,⊥ Jordi Juarez-Jimenez,‡,⊥ Cristobal de los Rıos, Mourad Chioua, Ramon Pouplana,
́
́
́
́
́
F. Javier Luque,‡ Mercedes Unzeta,*,† Jose Marco-Contelles,*,§ and Abdelouahid Samadi§
́
†
́
́
Departament de Bioquımica i Biologıa Molecular, Facultat de Medicina, Institut de Neurocien
̀
cies, Universitat Auto
̀noma de
Barcelona, 08193 Bellaterra, Barcelona, Spain
‡
́
Departament de Fisicoquımica, Facultat de Farmac
̀
ia, and Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal
643, E-08028, Barcelona, Spain
§
́
Laboratorio de Radicales Libres y Quımica Computacional (IQOG, CSIC), Juan de la Cierva 3, E-28006, Madrid, Spain
∥Instituto Teo
́
́
́
́
ica, Hospital Universitario de la Princesa, C/Diego de Leo
E-28029, Madrid, Spain
S
* Supporting Information
ABSTRACT: A new family of multitarget molecules able to interact with
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as
with monoamino oxidase (MAO) A and B, has been synthesized. Novel
compounds (3−9) have been designed using a conjunctive approach that
combines the benzylpiperidine moiety of the AChE inhibitor donepezil (1)
and the indolyl propargylamino moiety of the MAO inhibitor N-[(5-
benzyloxy-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine
(2), connected through an oligomethylene linker. The most promising
hybrid (5) is a potent inhibitor of both MAO-A (IC50 = 5.2 ± 1.1 nM) and
MAO-B (IC50 = 43 ± 8.0 nM) and is a moderately potent inhibitor of
AChE (IC50 = 0.35 ± 0.01 μM) and BuChE (IC50 = 0.46 ± 0.06 μM).
Moreover, molecular modeling and kinetic studies support the dual binding
site to AChE, which explains the inhibitory effect exerted on Aβ
aggregation. Overall, the results suggest that the new compounds are promising multitarget drug candidates with potential
impact for Alzheimer’s disease therapy.
Thus, monoamine oxidase (MAO, EC 1.4.3.4), the enzyme that
catalyzes the oxidative deamination of a variety of biogenic and
xenobiotic amines,13 is also an important target to be
considered for the treatment of specific features of this
multifactorial disease. MAO exists as two distinct enzymatic
isoforms, MAO-A and MAO-B, based on their substrate and
inhibitor specificities.14 MAO-A preferentially deaminates
serotonin, adrenaline, and noradrenaline and is selectively and
irreversibly inhibited by clorgyline. In contrast, MAO-B
preferentially deaminates β-phenylethylamine and benzylamine
and is irreversibly inhibited by R-(−)-deprenyl.15 Selective
inhibitors for MAO-A have been shown to be effective
antidepressants, whereas MAO-B inhibitors, although appa-
rently devoid of antidepressant action, are useful in the
INTRODUCTION
■
Alzheimer’s disease (AD), the most common form of adult
onset dementia, is an age-related neurodegenerative disorder
characterized by a progressive memory loss, a decline in
language skills, and other cognitive impairments.1 Although the
etiology of AD is not completely known, several factors such as
amyloid-β (Aβ)2 deposits, τ-protein aggregation,3 oxidative
stress,4,5 and low levels of acetylcholine (ACh) are thought to
play significant roles in the pathophysiology of the disease.6
The selective loss of cholinergic neurons in AD results in a
deficit of ACh in specific brain regions that mediate learning
and memory functions.7 However, alterations in other neuro-
transmitter systems, especially serotoninergic and dopaminer-
gic,8,9 are also thought to be responsible for the behavioral
disturbances observed in patients with AD.10 This evidence has
led to the suggestion that inhibitors of monoamine oxidase
(IMAOs) might be also valuable for the treatment of AD.11,12
Received: February 25, 2011
Published: October 24, 2011
© 2011 American Chemical Society
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treatment of Parkinson’s disease.16 Besides the increased amine
neurotransmission, the beneficial properties of IMAOs are also
related to the reduction of the formation of the neurotoxic
products, such as hydrogen peroxide and aldehydes, which
promote the formation of reactive oxygen species (ROS) and
may ultimately contribute to increased neuronal damage.17,18
Moreover, AD patients commonly present depressive symp-
toms that have even been considered as a risk factor for the
development of the disease.19 Increased MAO-B levels due to
enhanced astrogliosis in the brain of AD patients have also been
reported.11 Overall, these observations suggest that dual
inhibition of MAO-A and MAO-B, rather than MAO-B alone,
may be of value for AD therapy.
At present, there are three FDA-approved drugs (donepezil,
galanthamine, and rivastigmine)20−22 that improve AD
symptoms by inhibiting acetylcholinesterase (AChE, EC
1.1.1.7), i.e., the enzyme responsible for the hydrolysis of
ACh, and thereby raising ACh content in the synaptic cleft.
Apart from the beneficial palliative properties of AChE
inhibitors in AD,23−25 cholinergic drugs have shown little
efficacy to prevent the progression of the disease. In fact, the
multifactorial nature of AD supports the most current
innovative therapeutic approach based on the “one molecule,
multiple targets” paradigm.26,27 Thus, a single drug that acts on
a specific target to produce the desired clinical effects might not
be suitable for the complex nature of AD. Accordingly, the
multitarget-directed ligand (MTDL) approach has been the
subject of increasing attention by many research groups, which
have developed a variety of compounds acting on very diverse
targets.28−33 A very successful approach came from the
combination of the carbamate moiety of rivastigmine with the
indolamine moiety present in rasagiline, a well-known MAO-B
inhibitor, leading to the compound ladostigil.34 Besides
inhibiting MAO and AChE, it possesses neuroprotective and
antiapoptotic activities,35 which have been attributed to the
propargylamine group present in the molecule, thus retaining
the beneficial properties observed for rasagiline.36 The potential
therapeutic effect of this compound, which has reached clinical
trials,37 is also supported by recent findings showing the ability
of propargylamine-containing compounds to modulate cleavage
of β-amyloid protein precursor.38 Hybrid compounds targeting
cholinesterases and amyloid plaques,39 as well as site-activated
chelators targeting MAO and AChE, have also been recently
attempted.40−43
In the development of IMAOs for the treatment of
neurodegenerative diseases, our group initially extensively
investigated the effect of the introduction of a benzyloxy
group in a series of acetylenic and allenic derivatives of
tryptamine, which were previously reported to be selective for
MAO-A.44 We observed that the introduction of this moiety
changed the selectivity toward the B isoform of the enzyme and
that it was significantly decreased when a hydrogen atom was
attached to the nitrogen atom of the indole ring and/or the side
chain was substituted by a CH3 group.45,46 On the basis of
these previous works, we have designed a novel family of hybrid
compounds of type I to act as potential inhibitors of both MAO
and AChE (Figure 1). The novel hybrids have been conceived
by a conjuctive approach that combines donepezil (1) and N-
[(5-benzyloxy-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-
2-yn-1-amine (2), which is one of the most interesting IMAOs
previously investigated in our laboratory.46 The underlying
strategy is to retain the 1-benzylpiperidine fragment present in
donepezil (1), which binds to the catalytic and mid-gorge sites
Figure 1. Schematic representation of the conjunctive approach
designed to synthesize the novel IMAO/IAChE hybrids.
of AChE, with the 1-methyl-1H-indol-2-yl)methyl]-N-methyl-
prop-2-yn-1-amine moiety shown in compound 2 (Figure 1),
which should occupy the substrate binding site in MAO.
With this conjunctive approach, the novel hybrids are
expected to behave as dual binding site AChE inhibitors,
since the 1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-
1-amine moiety could presumably interact at the peripheral
anionic site (PAS) of AChE. The possibility of targeting both
catalytic active site (CAS) and PAS of AChE will largely
depend on the length of the linker, a crucial structural feature to
facilitate the binding of both 1-benzylpiperidine and [(1-
methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine
moieties to CAS and PAS, respectively, in AChE. This
particular mode of action should result in a significant AChE
inhibitory potency, of interest for the management of the
symptomathology of AD arising from the cholinergic deficit,
but more interestingly, it could also recognize the peripheral
site, which appears to mediate the Aβ proagreggating action of
AChE.47−50 On the other hand, the correct alignment of the 1-
benzylpiperidine and 1-methyl-1H-indol-2-yl)methyl]-N-meth-
ylprop-2-yn-1-amine moieties in MAO will also depend on the
tether, as the length and chemical nature of the linker should
also affect the accommodation of the hybrid through the
residues that define the bottleneck between the entrance and
substrate cavities in MAO.
In this work we describe the synthesis, pharmacological
evaluation, and molecular modeling of representative molecules
of this new family of compounds (I, Figure 1). The
pharmacological evaluation of these novel compounds includes
AChE and butyrylcholinesterase (BuChE) inhibition, the
inhibition of MAO-A and MAO-B, the kinetics of enzyme
inhibition, and the AChE-dependent and self-induced Aβ
aggregation. Finally, molecular modeling studies are performed
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a
to gain insight into the binding mode and structure−activity
relationships of the novel hybrid compounds.
Scheme 2
RESULTS AND DISCUSSION
Chemistry. To explore the suitability of the conjunctive
strategy outline above, compounds 3−9 (Figure 2) were
■
a
Reagents and conditions: (a) (EtO)2P(O)CH2CO2Et, THF, K2CO3,
reflux (92%); (b) (i) H2, Pd/C 10%, PtO2, 4 N HCI in dioxane,
EtOH, room temp; (ii) MeOH (90%); (c) BnBr, TEA, CH2CI2
(75%); (d) LiAIH4, THF, reflux (98%); (e) SOCI2, CH2CI2, reflux
(99%).
Figure 2. General structure for the target molecules 3−9.
synthesized, differing in the length of the tether and the
location and/or the number of nitrogens in the tethered-benzyl
substituted cyclohexane ring linked to the indolyl moiety.
The 1-benzyl 4-substituted piperidine derivatives 3−6 were
synthesized by sodium hydride/DMF promoted reaction of
compounds 10−13 and 1-methyl-2-{[ethyl(prop-2-yn-1-yl)-
amino]ethyl}-1H-indol-5-ol 1451 (Scheme 1).
a
Scheme 3
a
Scheme 1
a
Reagents and conditions: (a) (EtO)2P(O)CH2CHCHCO2Et,
EtOH, NaH, reflux (78%); (b) H2, Pd/C 10%, EtOH, room temp
(99%); (c) BnBr, TEA, CH2CI2 (70%); (d) LiAIH4, THF, reflux
(99%); (e) SOCI2, CH2CI2, reflux (99%).
piperidone 20 with triethyl 4-phosphonocrotonate in the
presence of sodium hydride in dry ethanol afforded compound
21 in 78% yield, whose catalytic hydrogenation over Pd/C in
ethanol at room temperature gave ester 22 in 99% yield.57
Next, reaction of 22 with benzyl bromide to give ester 23
followed by reduction with LAH gave the desired alcohol 24,
which was then treated with thionyl chloride to give the chloro
derivative 13 in almost quantitative yield.57
Compounds 7−9 (Figure 2) were synthesized as shown in
Schemes 4 and 5. Reaction of indole 14 with 1,2-dibromo-
ethane gave 25, which after treatment with 4-benzylpiperidine
afforded 7 (Scheme 4).51 Similarly, the reaction of indole 14
with 1,3-dibromopropane gave intermediate 26, whose reaction
with 4-benzylpiperidine or 1-benzylpiperazine afforded 8 and 9,
respectively (Scheme 5).51
a
Reagents and conditions: (a) NaH, DMF, room temp.
1-Benzyl-4-(chloromethyl)piperidine 10 and 1-benzyl-4-
(chloroethyl)piperidine 11 were synthesized following the
methods reported in the literature.52 1-Benzyl-4-(3-
chloropropyl)piperidine 12 was prepared as shown in Scheme
2, starting from commercial 4-pyridinecarboxaldehyde 15, via
the known intermediate 16,53 whose hydrogenation,54 under
Pd/C and PtO2, in the presence of hydrochloric acid and
workup with methanol afforded methyl ester 17.55 Next, N-
benzylation to give 1-benzylpiperidine 18, reduction with
lithium aluminum hydride (LAH) to provide alcohol 19,56 and
treatment with thionyl chloride furnished the chloride
derivative 12 in quantitative yield.
All new compounds showed analytical and spectroscopic data
in good agreement with their structures (see Experimental
Part).
1-Benzyl-4-(4-chlorobutyl)piperidine 13 was prepared as
shown in Scheme 3. Treatment of commercial 1-benzyl-4-
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a
Scheme 4
Table 1. AChE and BuChE Inhibitory Activities of
Donepezil (1), the Reference Compound 2, and N-[(1-
Methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amines
3−9
a
IC50 (μM)
selectivity
eqBuChE/EeAChE
compd
EeAChE
eqBuChE
1 (donepezil)
2
0.0067 ± 0.0004
>100
7.4 ± 0.10
>100
1100
>100
3.5
3 (n = 0)
4 (n = 1)
5 (n = 2)
6 (n = 3)
7 (n = 1)
8 (n = 2)
9 (n = 2)
0.31 ± 0.04
0.42 ± 0.04
0.35 ± 0.01
0.26 ± 0.07
>100
1.1 ± 0.20
2.1 ± 0.20
0.46 ± 0.06
0.99 ± 0.08
0.80 ± 0.10
2.2 ± 0.40
7.6 ± 0.40
5.0
1.3
3.8
a
Reagents and conditions: (a) Br(CH2)2Br, 2-butanone, K2CO3, 85
>100
0.12
>100
°C, 6 h (37%); (b) 4-benzylpiperidine, K2CO3) THF, 80 °C (77%).
18.1 ± 0.40
>100
a
Scheme 5
a
Values are expressed as the mean ± standard error of the mean of at
least three different experiments in quadruplicate.
in EeAChE. A drastic reduction in activity is also found upon
replacement of the piperidine by a piperazine unit (leading to
compound 9). Thus, compounds 7 and 9 are completely
inactive against EeAChE, whereas the inhibitory activity of 8 is
decreased 52-fold. Nevertheless, these chemical modifications
have less effect on the eqBuChE potency, as compounds 7−9
are roughly equipotent (7) or slightly less potent (8 and 9)
compared to 3−6.
Overall, the results point out the relevant role played by the
1-benzylpiperidin-4-yl unit on the AChE inhibitory activity,
suggesting that this moiety is the main factor in mediating the
binding to AChE. On the other hand, it is worth noting that
compounds 3−6 are active for the BuChE inhibition. This is
particularly important in view of the renewed interest in dual
AChE/BuChE cholinergic inhibitors as therapeutic agents for
AD, as they have been described to improve cognition.58 More
specifically, compound 5 is presented as a dual ChE inhibitor
by having both AChE and BuChE inhibition in the same
submicromolar level. For this reason and because of its
pharmacological properties, we evaluated its inhibitory activity
against human recombinant AChE (hrAChE), the cerebral
enzyme expressed in the HEK-293 cell line, and human serum
BuChE (hBuChE). Thus, compound 5 inhibited hrAChE with
an IC50 of 0.38 ± 0.05 μM (tacrine, used as a standard,
inhibited hrAChE with an IC50 of 122 ± 2 nM) and hBuChE
with an IC50 of 1.7 ± 0.2 μM (tacrine inhibited hBuChE with
an IC50 of 36 ± 4 nM).
Kinetic Studies. To gain further insight into the mechanism
of action of this family of compounds on AChE, a kinetic study
was carried out with the most promising compound of the
series, 5, using EeAChE. Graphical analysis of the reciprocal
Lineweaver−Burk plots (Figure 3) showed both increased
slopes (decreased Vmax) and intercepts (higher Km) at
increasing concentration of the inhibitor. This pattern indicates
a mixed-type inhibition and therefore supports the dual site
binding of this compound. Replots of the slope versus
concentration of compound 5 gave an estimate of the inhibition
constant, Ki, of 149 nM.
a
Reagents and conditions: (a) Br(CH2)3Br, 2-butanone, K2CO3, 85
°C, 6 h (80%); (b) 4-benzylpiperidine, K2CO3, THF, 80 °C (64%);
(c) 1-benzylpiperazine, K2CO3, THF, 80 °C (85%).
AChE and BuChE Inhibition. To study the multipotent
profile of the hybrid compounds, they were first evaluated as
inhibitors of AChE and BuChE. The AChE inhibitory activity
was tested against the Electrophorus electricus enzyme (Ee-
AChE), and the inhibition of BuChE was carried out using the
equine serum enzyme (eqBuChE). The inhibitory activities of
the hybrids were compared with those determined for the
parent compounds, donepezil (1) and N-[(5-benzyloxy-1-
methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine
(2).46
Biological Evaluation. The 1-benzylpiperidin-4-yl deriva-
tives 3−6 were found to be moderately potent regarding the
inhibition of EeAChE. IC50 values were similar in all cases
(ranging from 0.26 to 0.42 μM, Table 1). Moreover, they
exhibit similar potencies against eqBuChE, as IC50 values range
from 0.46 to 2.1 μM, leading to a very slight selectivity for
AChE (the ratio IC50(eqBuChE)/IC50(EeAChE) varies from
1.3 to 5.0). Accordingly, the length of the linker does not seem
to be a crucial factor for the inhibitory potency against AChE
and BuChE. The most potent compounds are 5 and 6, which
are characterized by IC50 values of 0.35 and 0.26 μM against
AChE and IC50 values of 0.46 and 0.99 μM against BuChE.
Compared with donepezil (1), they are, respectively, 52- and
39-fold less potent for the inhibition of AChE but 16- and 7-
fold more potent regarding the BuChE inhibition.
MAO Inhibition. To complete the study of the multipotent
biological profile of the hybrid compounds, the inhibitory
activity against MAO-A and MAO-B (from rat liver
mitochondria) was determined and compared with the
inhibition exerted by the parent compounds donepezil (1)
Compared to derivatives 3−6, reversion of the piperidine
ring in 7 and 8 has a dramatic effect on the inhibitory potency
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inhibitory potency but a lower activity toward MAO-B.
Interestingly, we found that compound 7 was 21-fold and 11-
fold less potent for MAO-A and MAO-B, respectively, than the
analogous inhibitor 4. Similarly, compounds 8 and 9 were
significantly less potent for both isoforms than the analogous
inhibitor 5 (12-fold and 6-fold for MAO-A and 256-fold and
38-fold for MAO-B, respectively). Altogether, these results
show that compounds bearing the donepezil 1-benzylpiperidin-
4-yl motif were the best MAO inhibitors and that, among them,
5 was the most potent inhibitor, even more than the reference
compound 2.
Kinetic Studies. To study the type of inhibition toward
MAO, we analyzed the reversibility/irreversibility of the
binding of compound 5, the most potent inhibitor of the
series. We observed that 5 irreversibly inhibited MAO-A, since
the inhibition was not reverted after three consecutive
centrifugations and washings with buffer (Figure 4A). This
hypothesis is also in agreement with the time-dependent
inhibition of MAO-A upon incubation with the inhibitor
(Figure 4B) and thus reflects the inhibition mechanism found
for the parent compound 2.46 Strikingly, whereas the inhibition
of MAO-B was also found to depend on the incubation time
(Figure 4D), a significant fraction (80.5 ± 3.3%) of the activity
was recovered by washing the enzyme three times after
incubation for 30 min (Figure 4C). These findings point out
that the addition of the benzylpiperidine unit to the reference
compound 2 in order to yield 5 does not affect the proper
alignment of the indolylpropargylamino moiety in the binding
cavity of MAO-A, thus leading to the complete inactivation of
the enzyme by chemical modification of the cofactor. However,
the present results suggest that docking of 5 into the binding
cavity of MAO-B is more impeded than in MAO-A, which
should prevent the proper alignment of the propargylamino
moiety, thus making less efficient the inactivation of the
enzyme.
To clarify the different behavior of 5 toward MAO-A and
MAO-B, we further investigated the progress curves of
substrate consumption for a longer period in the presence of
the inhibitor. As expected, the final rate became zero in both
cases, proving that the inhibition of 5 toward both MAO-A and
MAO-B occurs in an irreversible process. Nevertheless, the
time needed for the total inactivation of MAO-B was higher
than that needed for MAO-A (Figure 5), thus showing that
although irreversible, the inactivation of MAO-B by 5 is clearly
slower. These findings explain the different behavior found in
the reversibility studies, which were performed preincubating
the enzymes with 5 for 30 min. Figure 5 shows that at this time
the velocity of the reaction for MAO-A was clearly decreased
and thus substrate consumption was rapidly approaching zero.
In contrast, the velocity of the reaction for MAO-B shows a
little reduction at 30 min preincubation time, thus supporting
the suggestion that docking of 5 into the cavity of MAO-B is
more impeded than in MAO-A, resulting in a slower substrate
consumption and explaining the recovery of MAO-B activity in
the reversibility study.
Figure 3. Kinetic study on the mechanism of EeAChE inhibition by
compound 5. Overlaid Lineweaver−Burk reciprocal plots of AChE
initial velocity at increasing substrate concentration (0.1−1 mM) in
the absence of inhibitor and in the presence of 5 are shown. Lines were
derived from a weighted least-squares analysis of the data points.
and N-[(5-benzyloxy-1-methyl-1H-indol-2-yl)methyl]-N-meth-
ylprop-2-yn-1-amine (2).
Biological Evaluation. The 1-benzylpiperidin-4-yl deriva-
tives 3−6 showed a potent MAO-A inhibition (Table 2), acting
Table 2. MAO-A and MAO-B Inhibitory Activities of
Donepezil (1), Reference Compound 2, and the N-[(1-
Methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine
Derivatives (3−9)
a
IC50 (nM)
selectivity
MAO-B/MAO-A
compd
MAO-A
MAO-B
1 (donepezil)
2
850000 ± 13000
100 ± 7.0
82 ± 3.0
6.7 ± 1.8
5.2 ± 1.1
10 ± 4.0
140 ± 44
65 ± 17
31 ± 14
15000 ± 2200
63 ± 2.0
750 ± 20
130 ± 41
43 ± 8.0
2700 ± 110
1400 ± 500
11000 ± 2400
1600 ± 710
0.020
0.63
9.1
3 (n = 0)
4 (n = 1)
5 (n = 2)
6 (n = 3)
7 (n = 1)
8 (n = 2)
9 (n = 2)
19
8.3
260
10
170
54
a
Values are expressed as the mean ± standard error of the mean of at
least three different experiments in quadruplicate.
in the nanomolar range (IC50 ranging from 82 ± 3.0 to 5.2 ±
1.1 nM). In contrast, they were less potent against MAO-B,
with the exception of 5, which was found to be the most potent
compound toward both isoforms (IC50 of 5.2 ± 1.1 and 43 ±
8.0 nM for MAO-A and MAO-B, respectively). The most
selective inhibitor was compound 6 (n = 3) toward MAO-A,
whereas 5 was much less selective. It is worth noting the large
sensitivity of the inhibitory potency on the length of the tether.
Thus, removal of a single methylene group in 6 to yield 5
increased the inhibitory potency against MAO-A and MAO-B
by a factor of 2 and 63, respectively. Likewise, further reduction
of the tether to just one methylene (from 5 to yield 4) did not
affect the MAO-A inhibitory potency but reduced the potency
against MAO-B by 3-fold.
The kinetic behavior of 5 toward MAO-B determined from
the initial rates showed that this compound behaves as a
noncompetitive inhibitor, as shown in the Lineweaver−Burk
plot (Figure 6A). Thus, the Vmax decreased as the amount of 5
was increased, whereas the KM value remained constant (Figure
6B). Determination of Michaelis constants gave a value of KM =
6.7 ± 0.3 μM and Vmax = 277.8 ± 6.1 pmol/min for MAO-B
Compounds 7 and 8, bearing a 4-benzylpiperidin-1-yl
residue, also inhibited MAO-A quite potently but showed less
potency toward MAO-B. Similarly, compound 9, containing a
4-benzylpiperazin-1-yl residue, also showed a good MAO-A
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Figure 4. Reversibility studies of MAO-A and MAO-B inhibition by compound 5. MAO-A (A) and MAO-B (C) were inhibited at 6 and 45 nM 5,
respectively, for 30 min. Then three consecutive washings were performed with buffer. MAO-B inhibition (C) was reverted (recovering 80.5 ± 3.3%
of enzyme activity), whereas the inhibition of MAO-A remained unaltered after washings (A). Time-dependence inhibition was studied at several
times of preincubation (0−30 min) of MAO with compound 5. Both MAO-A (B) and MAO-B (D) inhibition were found to be time-dependent.
Data are the mean ± SEM of four independent experiments in triplicate.
and MAO-B. Moreover, reversion of the piperidine unit in
compounds 5 and 8 notably affects the inhibition of both AChE
and MAO. Finally, 5 leads to an irreversible inhibition of MAO-
A, whereas inactivation of MAO-B is slower. To shed light onto
these questions, a series of docking and molecular dynamics
(MD) simulations were conducted to identify the binding
mode of 5 to AChE, BuChE, MAO-A, and MAO-B.
AChE Inhibition. The binding mode of 5 to AChE was
investigated by considering three structural models of the
enzyme, which were built up taking advantage of the X-ray
structure of donepezil bound to Torpedo californica AChE
(TcAChE, PDB entry 1EVE).59 These models retain the
Figure 5. Progress curves of substrate consumption for MAO-A (5-
HT, 100 μM) and MAO-B (PEA, 20 μM) in the presence of 5 (10 nM
for MAO-A and 100 nM for MAO-B). MAO-A and MAO-B were
preincubated with the inhibitor for a long period (0−420 min), and
the catalytic activity was determined. The time needed for the total
inactivation of the enzyme was greater for MAO-B than for MAO-A.
Data are the mean ± SEM of three independent experiments in
triplicate.
structural details of the benzylpiperidine moiety bound to the
AChE gorge but differ in the orientation of Trp279 (numbering
in TcAChE), as the inspection of the available X-ray structures
for complexes of AChE with dual site binding inhibitors reveals
that Trp279 adopts three main conformations at the PAS.60,61
Thus, the side chain of Trp279 can be characterized by dihedral
angles χ1 (N−Cα−Cβ−Cγ) and χ2 (Cα−Cβ−Cγ−Cδ2) close to
(i) −60° and −80°, (ii) −120° and +50°, and (iii) −160° and
−120°. Representative structures of these orientations are PDB
entries 1EVE, 2CKM, and 1Q83, which correspond to the
AChE complexes with donepezil, bis(7)-tacrine,62 and syn-
TZ2PA6,63 respectively. In the following these models will be
denoted AChE(1EVE), AChE(2CKM) and AChE(1Q83).
The binding of 5 to the three AChE models was first
explored by docking calculations performed with rDock,64,65
which yielded reliable predictions for the binding mode of
substrate, β-phenylethylamine (PEA), and an estimated Ki of
11.0 ± 0.39 nM.
Molecular Modeling. The preceding studies point out
that 5 seems to be a promising multitarget inhibitor. However,
they also show distinctive trends in the pharmacological profile.
First, it is unclear why the inhibitory potency against AChE
(and BuChE) is slightly affected by the length of the tether,
whereas it has a large effect in the inhibition of both MAO-A
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Figure 6. Kinetic study on the mechanism of MAO-B inhibition by 5. (A) Overlaid Lineweaver−Burk reciprocal plots of MAO-B initial velocity at
increasing substrate concentration (PEA, 1−200 μM) in the absence or presence of 5 (10−100 nM) are shown. Lines were derived from a weighted
least-squares analysis of the data points. (B) Vmax decreased as the amount of 5 increased, whereas the KM value remained constant. Data are the
mean ± SEM of four independent experiments in triplicate.
known dual site binding AChE inhibitors.60 Suitable restraints
were introduced to impose the benzylpiperidine moiety to
mimic the known binding mode of the same fragment of
donepezil in its complex with TcAChE.59 In turn, this permits
enhancement of the conformational sampling of the
indolylpropargylamino moiety at the PAS. Finally, the structural
integrity and energetic stability of the binding mode proposed
for each AChE−5 complex were examined from the snapshots
sampled in 20 ns MD simulations. For the sake of comparison,
an additional 20 ns MD simulation was run for the complex
between AChE and donepezil.
MD simulations yielded stable trajectories in the last 10 ns, as
noted by the time evolution of the potential energy and the
root-mean-square deviation (rmsd) of the protein backbone,
which ranged from 1.4 to 1.6 Å (a rmsd value of 1.3 Å was
obtained for the AChE−donepezil complex; see Figures S1 and
S2 in Supporting Information). The rmsd values of the residues
that delineate the binding site in catalytic, mid-gorge, and
peripheral sites (defined as those residues with at least one
atom placed at a distance less than 4 Å from the ligand) were
stable for AChE−donepezil (1.4 Å) and AChE(1EVE)−5 (1.6
Å) complexes, they being similar to the rmsd values determined
for the backbone. Larger rmsd values were found for the
binding site residues in AChE(2CKM) (2.0 Å) and AChE-
(1Q83) (1.7 Å).
No large structural alterations were found in the catalytic site,
Figure 7. Representation of the binding mode of donepezil and 5 at
the beginning and end of the MD simulations: (top left) AChE−
donepezil (started from the X-ray structure in PDB entry 1EVE); (top
right) AChE(1EVE)−5; (bottom left) AChE(1Q83)−5; (bottom-
right) AChE(2CKM)−5. Relevant residues at the catalytic (Trp84,
Phe330) and peripheral (Trp279, Tyr70) binding sites are also shown.
The ligand/residues at the end of the simulations are shown in blue/
green, respectively (the representation at the beginning of the
simulation is made in orange/yellow).
and the benzylpiperidine moiety adopts a similar arrangement
in all cases (Figure 7). Little structural fluctuations were also
found in the mid-gorge and peripheral sites for complexes
AChE−donepezil and AChE(1EVE)−5. In particular, the
indanone unit of donepezil and the indolyl ring of 5 were
stacked against Trp279, whose side chain was stable along the
trajectory. Thus, the torsional angles χ1 and χ2 showed small
fluctuations around average values of −70° and −110° (see
Figure S3 in Supporting Information; the corresponding angles
in 1EVE are −52° and −84°). In contrast, larger rearrange-
ments were found at the PAS, mainly involving Trp279, for
AChE(1Q83)−5 and AChE(2CKM)−5. In the former case χ1,
which was initially assigned a value close to 180° (as found in
1Q83), remained stable during the first 11 ns but then suddenly
changed to a value close to −60° (see Figure S3). Thus, the
initial binding mode, which was chosen to mimic the
orientation of syn-TZ2PA6 bound to TcAChE, was lost and
Trp279 adopted a new arrangement close to the conformation
found in the AChE−donepezil and AChE(1EVE)−5 complexes
(see above and Figure 7). With regard to AChE(2CKM)−5,
Trp279 was initially oriented to reproduce the conformation
found in the TcAChE−bis(7)-tacrine complex (characterized
by χ1 and χ2 close to −120° and +50°). After 3 ns, the angle χ1
changed to 180° and remained stable for 3 ns but then changed
to a new value of +60°, which was stable along the rest of the
trajectory (see Figure S3 in Supporting Information). Such a
change was accompanied by the readjustment of χ2, which
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adopted a value close to +90°. At the end of the simulation, a
novel structure was obtained where the protonated propargy-
lamino unit remains close to Tyr70 but has lost the interactions
with Trp279 (see Figure 7).
largest structural flexibility of the inhibitor is localized in the
propargylamino unit, which forms transient van der Waals
interactions with the benzene ring of Tyr70.
This binding mode allows us to rationalize the low sensitivity
of the inhibitory activity on the length of the tether, as (i) it is
reasonable to expect that the benzylpiperidine moiety will fill
the same binding pocket but (ii) shortening or lengthening of
the tether will be accompanied by displacements of the indole
ring of 5 along the gorge that would retain the stacking against
Tyr334 or Trp279 (see Figure S5 in Supporting Information).
On the other, the lower inhibitory potency associated with
reversion of the piperidine unit (compounds 5 and 8) can be
explained by two factors: (i) the lost of the water-assisted
hydrogen bonds formed between the protonated nitrogen and
Tyr121 and Ser122 (data not shown) and (ii) the weakening of
the electrostatic stabilization due to cation−π interactions with
the aromatic rings of Phe330 and Trp84 and with the negative
charges of Asp72 and Glu198 (see Figure S6).
The preceding analysis demonstrates the structural integrity
of the AChE(1EVE)−5 model, which mimics the structural
features of the binding of donepezil to TcAChE. The reliability
of this binding mode is reinforced by the conformational
change observed in the PAS of the AChE(1Q83)−5 complex,
which leads to a binding mode close to that found in
AChE(1EVE)−5, and by the structural change found in
AChE(2CKM)−5, which leads to a binding mode characterized
by a conformation of Trp279 that, to the best of our
knowledge, has no counterpart in the available X-ray structures
of AChE complexes deposited in the PDB. As a further test, we
have determined the relative binding affinity between models
AChE(1EVE)−5 and AChE(2CKM)−5 by means of MM/
PBSA calculations. They were performed for 100 snapshots
taken evenly during the last 5 ns of the trajectories using both
the standard radii implemented in the AMBER force field and
an alternative set of atomic radii that have been explicitly
optimized for their use in MM/PBSA calculations with
AMBER.66 The results (see Table S1 in Supporting
Information) indicate that binding of 5 to AChE(1EVE) is
favored by near 2.3 (standard radii) and 4.0 (optimized radii)
kcal/mol relative to AChE(2CKM). Similar trends were
observed when MM/PBSA computations were performed by
retaining a single water molecule that was hydrogen-bonded to
the protonated nitrogen of the piperidine unit of the ligand
along the trajectories (data not shown).
The preceding structural and energetic analysis suggests that
compound 5 mimics the binding mode of donepezil (see Figure
S4). Thus, the benzylpiperidine moieties of donepezil and 5
exhibit a similar alignment in the CAS, though there is a weaker
overlap between the benzene unit of 5 and the indole ring of
Trp84. More importantly, there is a change in the orientation of
residues Tyr334 and Asp72, which remain hydrogen-bonded
but are displaced toward the PAS. In turn, this structural change
facilitates the stacking of the indole ring of 5 between the
aromatic rings of Tyr334 and Trp279. On the other hand, there
is a water molecule that bridges the protonated nitrogen of the
piperidine unit with the hydroxyl groups of Tyr121 and Ser122
(not shown in Figure 8 for the sake of clarity). Finally, the
As a final test, we explored the suitability of an alternative
binding mode where the orientation of 5 was reverted so that
the propargylamino group fills the CAS and the benzylpiper-
idine moiety occupies the PAS. As in the preceding discussion,
compound 5 was docked in the binding site of the enzyme for
the three AChE structural models (1EVE, 1Q83, and 2CKM)
but without imposing positional restraints. Inspection of the
first ranked poses showed a preference for the placement of the
benzylpiperidine moiety in the CAS. Thus, only 1, 7, and 2
poses out of the first 13, 15, and 7 ranked solutions (covering a
range of 8 kcal/mol in the score in each case) corresponded to
the reverted orientation of 5 upon docking to models
AChE_1EVE, AChE_1Q83 and AChE_2CKM, respectively,
which reinforces the reliability of the binding mode discussed
above. To further explore the suitability of the inverted binding
mode, a series of 20 ns MD simulations were run for the three
AChE complexes with the reversed orientation of the inhibitor.
Inspection of the binding mode sampled along the last 10 ns of
the trajectory run for AChE_1EVE (Figure S7) shows that the
propargylamino unit remains stacked onto the indole ring of
Trp84, filling part of the region occupied by the benzyl moiety
of donepezil. However, there are notable structural fluctuations
of the methylated indole ring of 5, which eventually leads to
steric clashes with the benzene ring of Phe330. Similarly, the
large fluctuations of the benzylpiperidine moiety also argue
against a firm stacking with Trp279 at the PAS. The structural
instability of the ligand was also found in the simulations run
for AChE_1Q83 and AChE_2CKM (see Figure S7),
particularly seen in the large mobility of the ligand at the
PAS, which affects the stacking between Trp279 and Tyr70. In
fact, comparison of the relative free energies determined from
MM/PBSA calculations for the different AChE complexes also
supports the energetic destabilization of the inverted binding
mode (see Table S2). Finally, let us remark that the enhanced
flexibility of the piperidine moiety in the PAS, which reflects the
lack of strong interactions, does not provide a straightforward
explanation to the significant reduction in the inhibitory
potency found upon reversion of the ring (compare 5 and 8 in
Table 1).
BuChE Inhibition. The binding mode of 5 to BuChE was
explored by means of docking calculations (see Experimental
Part). The results indicate a marked preference for the insertion
of the benzylpiperidine moiety in the CAS, as noted by the fact
that only 4 out of the first 20 ranked poses (comprising a range
of 5 kcal/mol in the score) were found with the inverted
Figure 8. Representation of the binding mode of 5 in (top) MAO-A
and (bottom) MAO-B. Five snapshots taken every ns along the last 5
ns of the trajectory are superposed. The ligand is shown as orange
sticks, FAD as blue sticks, and selected residues in the entrance and
substrate cavities as green sticks.
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arrangement (in fact, the first inverted pose was ranked as the
ninth solution). Hence, a new docking calculation was run
imposing the benzene unit of 5 to stack against Trp79
(equivalent to Trp84 in the CAS of AChE), thus mimicking the
interaction found in the AChE complex with donepezil (PDB
entry 1EVE). Even in this case, the results indicate a substantial
degree of flexibility to accommodate 5 in the binding site of
BuChE, especially regarding the indolylpropargylamine moiety
but also even the piperidine ring (see Figure S8). Nevertheless,
this finding is not unexpected because of the wider volume of
the binding site in BuChE compared to AChE, which can be
ascribed to mutations between specific binding site residues in
the two enzymes, such as the replacement of Phe330 in the
CAS of AChE by Ala, the substitution of Tyr70 and Phe290 at
the mid-gorge of AChE by Asn and Val, respectively, and the
mutation of Trp279 in the PAS of AChE by Ala. Accordingly, it
can be expected that the binding of 5 to BuChE will be mainly
guided by the interactions due to the benzylpiperidine moiety,
thus explaining the lack of large differences in the selectivity
between the two enzymes.
MAO Inhibition. The binding mode of hybrid 5 in MAO-A
and MAO-B was investigated in order to explain the different
inhibitory behavior found for the two isoforms. To this end, we
first explored the potential binding mode of 5 by means of
docking calculations, which showed a clear preference to
accommodate the indolylpropargylamino unit in the substrate
cavity. In fact, this is not surprising, as the reference compound
2 was found to be an irreversible inhibitor of the two MAO
isoforms, which indicates that the binding mode places the
propargylamino unit properly oriented to react with the flavin
adenine dinucleotide (FAD) present in the substrate cavity of
both MAO-A and MAO-B.43 Then a representative member of
the most populated cluster of the docked poses was chosen as
starting structure for MD simulations of the complexes of 5
with MAO-A and MAO-B. In the two cases MD simulations
yielded stable trajectories, as noted by inspection of the time
evolution of the potential energy and by the small fluctuations
of the rmsd profile along the last 5 ns of the trajectories (see
Figures S9 and S10). Thus, the rmsd determined for the
protein backbone in the MD simulations run for MAO-A and
MAO-B is close to 1.4 Å, whereas the rmsd determined for the
residues that define the walls of the binding cavity amounts to
2.0 Å.
of the ligand. On the other hand, simulations also show that the
positive charge of the piperidine unit in 5 is stabilized by water-
mediated hydrogen bond interactions with the backbone
carbonyl groups of Arg109 and Gly110.
The structural integrity of the binding mode of 5 in MAO-B
is also supported by inspection of the snapshots collected at the
end of the trajectory (Figure 8). The five-membered ring of the
indole moiety of 5 superposes the benzene ring of deprenyl in
its complex with human MAO-B (PDB entry 2BYB;67 see
Figure S12). As in MAO-A, the tether occupies the hydro-
phobic region delineated by residues Ile199, Ile316, Tyr326,
and Leu88, which presumably would lead to steric clases with
the piperidine ring upon shortening of the methylenic chain of
the inhibitor. Finally, besides water-mediated contacts with the
carbonyl groups of Arg100 and Gly101, the positive charge of
the piperidine unit in 5 appears to be stabilized by the
carboxylate group of Glu84.
As noted above, treatment of MAO-A with 5 leads to a
significant inactivation of the enzyme, which remains unaltered
after repeated washings. In contrast, a significant recovery of
MAO-B activity is found after washings because of a slower
inactivation of this isoform. This different behavior suggests
that the propargylamino moiety of 5 in MAO-A is better
oriented for chemical modification of FAD than in MAO-B.
Nevertheless, since both ligand and FAD are treated classically
in MD simulations, the electronic effects that promote the
chemical inactivation of the enzyme by the propargyl moiety
are not properly accounted for. Therefore, we have compared
the relative orientation of 5 obtained from MD simulations
with the orientation found for clorgyline, deprenyl, rasagiline,
and its ((ethyl(methyl)amino)carbonyl)oxy derivative (PDB
entries 2BXR, 2BYB, 1S2Q, and 2C65),67−69 paying attention
to the relative positioning of the carbon atom attached to the
protonated amine of 5 relative to the nitrogen atom of FAD
involved in chemical modification by the irreversible inhibitors.
Inspection of Figure 9 shows that in MAO-A such carbon atom
is slightly closer to the FAD nitrogen atom than in MAO-B.
Thus, the distance between those atoms (averaged over the
snapshots sampled in the last 5 ns) amounts to 6.8 ± 0.3 Å in
MAO-A and to 7.7 ± 0.4 Å in MAO-B (see Figure S13). The
larger separation found in MAO-B agrees with the lower degree
of enzyme inactivation found upon incubation with 5.
To further check this assumption, we have run additional
simulations forcing the nitrogen atom of the propargylamino
group to occupy the position and orientation relative to FAD
found for the corresponding atom in complexes of MAO-A
with clorgyline, and MAO-B with deprenyl, rasagiline, and its
((ethyl(methyl)amino)carbonyl)oxy derivative (structurally
related to ladostigil). Noteworthy, superposition of the X-ray
structures of those complexes reveals that the nitrogen atom of
the inhibitor occupies the same spatial location in the binding
site, as expected from the similar geometrical arrangement of
the propargylamino unit after chemical reaction with FAD (see
Figure 9). To this end, restrained simulations (1 ns) were run
in triplicate for each enzyme in order to force compound 5 to
occupy the position expected after chemical reaction with FAD.
With the aim to avoid artifactual results arising from steric
clashes of the propargylamino group with binding site residues
while steering the nitrogen atom, a methyl unit was used to
replace the propargyl moiety. Finally, we have determined the
difference in the interaction energy (from MM/PBSA
calculations) between the inhibitor and the enzyme.
Inspection of Figure 8 clearly shows that binding of 5 to
MAO-A has little effect on the residues that delineate the
binding site, suggesting a suitable fit of the indolylpropargyla-
mino unit in the substrate cavity. In fact, the indole fragment of
5 matches well the corresponding moiety in harmine, as noted
upon superposition of the X-ray structure of the human MAO-
A−harmine complex67 and the last snapshot of the MD
simulation (see Figure S11). The only exception to the
structural integrity of the binding pocket concerns few residues
located at the entrance of the gorge leading to the substrate
cavity, which reflects the adjustment of the ligand, as expected
from the larger flexibility of the solvent-exposed loops that
shape the entrance cavity (see Figure S11). It is worth noting
how the tether fills the hydrophobic region that defines the
bottleneck of the binding pocket, which is mainly due to
Phe208 and Ile325. The dependence of the inhibitory potency
with the length of the tether can be explained by the steric
contraints imposed by the side chains of vicinal apolar residues,
such as Leu97, Leu337, Val210, and Cys323, as shortening of
the tether would lead to steric clashes with the piperidine unit
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Finally, additional 20 ns MD simulations were run to
simulate the adduct of 5 covalently bound to FAD. In the two
cases MD simulations yielded stable trajectories, as noted by
inspection of the time evolution of the potential energy (see
Figure S14) and by the small fluctuations of the rmsd profile
along the last 5 ns of the trajectories (see Figure 11). However,
Figure 9. Superposition of the X-ray crystallographic structures of
MAO-A complexed with clorgyline (white, PDB entry 2BXR) and
MAO-B complexed with deprenyl (magenta, PDB entry 2BYB),
rasagiline (blue, PDB entry 1S2Q), and its ((ethyl(methyl)amino)-
carbonyl)oxy derivative (yellow, PDB entry 2C65). The spheres show
the position of the carbon atom that bears the protonated amine of 5
(orange) and the chemically modified nitrogen atom of FAD (blue).
Representation includes the positions of those atoms in snapshots
taken every ns along the last 5 ns of the trajectory run for the complex
(top) MAO-A−5 and (bottom) MAO-B−5.
Figure 10 shows the time evolution of the distance between
the ligand carbon atom (linked to the amino nitrogen) and the
Figure 11. Time evolution of the rmsd determined for the backbone
atoms (black) and the heavy atoms of the residues that define the
binding site (red) along the trajectories run for the (top) MAO-A−5
adduct and (bottom) MAO-A−5 adduct.
it is also worth stressing the different profile of the rmsd along
the whole trajectory. Thus, whereas there is a slight increase in
the rmsd determined for both the backbone atoms and the
heavy atoms of the residues that define the binding site in the
simulation run for the MAO-A−5 adduct (the rmsd increases
from 1 to 1.5 Å; see Figure 11), there is a more abrupt increase
in the rmsd determined for the binding site residues in the
simulation run for the MAO-B−5 adduct (the rmsd increases
from 1 to 2.2 Å; see Figure 11). This trend agrees with the
results derived from the restrained MD simulations discussed
above and indicates that the proper accommodation of the
inhibitor in a conformation suitable for covalent attachment to
FAD requires a larger rearrangement in the binding site of
MAO-B.
The different trends regarding the inhibitory activity of 5 in
MAO-A and MAO-B can be mainly ascribed to the residues
that define the bottleneck in the gorge that leads to the
substrate binding site. In particular, the replacement of Ile199
in MAO-B by Phe208 in MAO-A pushes the inhibitor toward
the FAD, whereas the replacement of Ile325 in MAO-A by
Tyr326 in MAO-B triggers the opposite effect. Thus, even
though there is a slight net stabilization in the interaction
energy of the inhibitor covalently bound to FAD in both MAO-
Figure 10. Time evolution of the distance between the ligand carbon
atom (linked to the amino nitrogen) and the nitrogen atom of FAD
along the restrained MD simulation (shown in dashed lines; MAO-A
in black and MAO-B in red). The change in the interaction energy
(estimated from MM/PBSA calculations) is shown by solid lines
(MAO-A in black and MAO-B in red). For clarity of comparison, the
interaction energy profiles are shown relative to the initial snapshot.
nitrogen atom of FAD along the restrained trajectories, and the
corresponding change in the ligand−receptor interaction free
energy estimated from MM/PBSA computations. The re-
strained simulations bring the nitrogen atom of 5 to the
position expected for the covalent adduct with FAD (distance
N(inhibitor)···N(FAD) at around 5.0 Å), the attached carbon
atom being located at around 6.4 Å. Interestingly, the results
indicate that the interaction energy in MAO-A becomes more
negative (relative to the initial state) at around 7 kcal/mol,
whereas it is destabilized at around 6 kcal/mol in MAO-B.
Therefore, from a qualitative point of view, these results
reinforce the notion that adoption of a reactive configuration of
5 in MAO-B is more impeded than in MAO-A, which agrees
with the different rates of enzyme inactivation.
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A and MAO-B (relative to the noncovalent form; see Table
S3), present results suggest that there is a larger barrier for the
proper accommodation of 5 in MAO-B. In this context, though
the methylenic (n = 2) tether designed for 5 seems well suited
to fit the gorge in the two isoforms, the spatial constraints
imposed on the orientation of the indole ring upon binding to
MAO-B leads to a less effective chemical interaction with the
FAD. In turn, these findings suggest that extension of the
((methyl)amino)propargylamino unit in 5 could be a useful
strategy to enhance the inactivation of MAO-B while reducing
the inhibition at MAO-A. This strategy could be beneficial to
minimize side effects related to the potentiation of the
cardiovascular effect of tyramine (the so-called “cheese effect”),
a limited side effect of older generation of nonselective MAO
inhibitors.
Inhibition of Aβ Self-Aggregation and AChE-Induced
Aggregation. A number of dual binding site AChE inhibitors
have been found to exhibit a significant inhibitory activity on
Aβ self-aggregation.60,70−75 Thus, compound 5 was also tested
for its ability to inhibit the self-induced Aβ1−42 aggregation and
the AChE-induced Aβ1−42 aggregation. In the former case a
47.8 ± 2.1% inhibition was found when compound 5 was tested
at 10 μM (Table 3) (concentration ratio Aβ/inhibitor = 4/1).
interact with the active, peripheral, and mid-gorge binding sites
of AChE, as well as to occupy the substrate binding site in
MAO.
The length of the tether that connects the two main
structural fragments of the novel hybrids has a relevant effect
on the binding to MAO, whereas it seems to have little impact
on the inhibitory activity against AChE and BuChE. Among
these hybrid compounds, 5 is the most potent IMAO, even
more than the parent compounds. Surprisingly, although
donepezil (1) is a slight inhibitor of BuChE and 2 is not
even active, the ability of 5 to inhibit BuChE is found on the
submicromolar range. This is particularly important in view of
the renewed interest in dual cholinergic inhibitors as
therapeutic agents for AD.58 Normally, AChE predominates
in the brain, while BuChE activity levels are low. However, in
AD the relative enzymatic activity is altered such that BuChE
increases while AChE decreases.77,78 Then, if the therapeutic
goal is to increase ACh levels in the brain, a compound able to
inhibit both AChE and BuChE would be valuable to elicit a
larger protective response. In addition, the inhibition of MAO-
B by 5 might be beneficial for modulating the cholinergic
neurotransmission and for restoring the serotoninergic neuro-
transmission. Moreover, the potent MAO-A inhibition enables
the drug to exert an antidepressant activity like that of
amitriptyline and moclobemide, two triciclic antidepressants
primarily used to treat depression. Compound 5 also presents a
significant inhibitory profile of Aβ-self-induced and human
AChE-dependent aggregation, being more potent (human
AChE-dependent) than or similar (self-induced) to the parent
compound donepezil (1). Overall, the present data indicate that
5 not only is an interesting lead compound for the design of
novel MTDL with a good IMAO/IAChE inhibitory potency
and a significant activity against amyloid aggregation but also
may have a potential disease-modifying role in the treatment of
AD.
Table 3. Inhibition of AChE-Induced Aβ1‑40 Aggregation and
Aβ1‑42 Self-Induced Aggregation by Compound 5
% inhibition of Aβ aggregation ± SEM
ac
,
bc
,
compd
AChE-induced
self-induced
47.8 ± 2.1
5
32.4 ± 7.0
a
Inhibition of AChE-induced Aβ1−40 aggregation. The concentration
of 5 and Aβ1−40 was 100 and 40 μM, respectively. The ratio Aβ/AChE
b
was equal to 100/1. Inhibition of self-induced Aβ1−42 aggregation (40
c
μM) produced by 5 at 10 μM. Data are the mean ± SEM of at least
three independent experiments.
We used propidium iodide (PI) as reference compound, and
we obtained a reduction of Aβ self-induced aggregation of 33.3
± 2.1%, this value being significantly lower than that found for
5. When PI was tested at equimolar concentrations (Aβ/PI =
1/1), similar to that previously reported by other groups, we
found a reduction of Aβ aggregation of 78.6 ± 3.8%. This effect
is significantly higher than that reported for donepezil (<5%)
under similar experimental conditions72 and similar to that
found for other IAChE.60,71,72 Then 5 can be considered a
moderate inhibitor of Aβ1−42 self-induced aggregation. Regard-
ing the inhibition of the human AChE-dependent Aβ1−40
aggregation, the results indicate that 5 at 100 μM was able to
prevent hAChE-induced Aβ1−40 aggregation in a 32.4 ± 7.0%
(Table 3). This value is similar to the inhibition elicited by
donepezil (22%) and significantly higher than that found for
tacrine (7%), two of the first FDA-approved drugs for the
treatment of AD. However, it is significantly lower than that of
propidium76 and other IAChEs previously described,70−72
which show potencies in the low micromolar range.
EXPERIMENTAL PART
■
General Methods. Melting points were determined in a Koffler
1
apparatus and are uncorrected. H NMR and 13C NMR spectra were
recorded at room temperature in CDCl3 or DMSO-d6 at 300, 400, or
500 MHz and at 75.4, 100.6, or 125.6 MHz, respectively, using solvent
peaks [CDCl3, 7.27 (D), 77.2 (C) ppm; DMSO-d6, 2.50 (D) and 39.7
(C) ppm] as internal references. The assignment of chemical shifts is
based on standard NMR experiments (1H, 13C, 1H−1H COSY,
1H−13C HSQC, HMBC, DEPT). Mass spectra were recorded on a
GC/MS spectrometer with an API-ES ionization source. TLC was
performed on silica F254. Detection was by UV light at 254 nm or by
spraying with phosphomolybdic-H2SO4 dye reagent. Column
chromatographies were performed on silica gel 60 (230 mesh).
“Chromatotron” separations were performed on a Harrison Research
model 7924. The circular disks were coated with Kieselgel 60 PF254
(E. Merck). The chlorydrate salts were prepared by solubilizing the
compound in a minimum of ether, and a solution of ether saturated
with HCl(g) was added dropwise. A white solid was formed
immediately. The precipitated hydrochloride was separated by
filtration, washed with ether, and dried. The purity (≥95%) of the
samples was determined by elemental analysis, carried out at the
IQOG (CSIC, Spain).
CONCLUSIONS
■
Methyl 4-(Piperidin-4-yl)butanoate (17).55 A solution of (E)-
ethyl 3-(pyridin-4-yl)acrylate 1652 (1.52 g, 8.58 mmol) in EtOH (20
mL) and 4 N HCl in 1,4-dioxane (2 mL) was hydrogenated under
10% Pd/C (0.152 g) and PtO2 (152 mg) overnight at room
temperature. Then the catalyst was filtered off, washed with MeOH,
and the filtrate was concentrated. The residual solid was triturated with
Et2O, filtered, washed with Et2O, and dried to give the known methyl
A new series of hybrid compounds containing the benzylpiper-
idine moiety of donepezil and the indolylpropargylamino
moiety of N-[(5-benzyloxy-1-methyl-1H-indol-2-yl)methyl]-N-
methylprop-2-yn-1-amine have been investigated as novel
multitarget agents against AChE, BuChE, and MAO (A/B).
These new compounds have been designed to simultaneously
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1
ester 17 (1.60 g, 90%) as a solid. H NMR (300 MHz, DMSO-d6) δ
32.8 (CH2), 36.2 (CH2), 45.0 (CH2Cl), 53.8 (2CH2), 63.4 (CH2-Ph),
126.8 (CH-Ph), 128.0 (2 × CH-Ph), 129.1 (2 × CH-Ph), 138.3 (C-
Ph); MS (EI) m/z (%) 91 (100) [PhCH2]+, 174 (42) [M −
(CH2)4Cl]+, 188 (43) [M − (CH2)3Cl]+, 202 (16) [M − (CH2)2Cl]+,
216 (8) [M − CH2Cl]+, 230 (28) [M − Cl]+, 264 (45) [M]+. HRMS
(ES+), exact mass calcd for C16H25ClN (M + H)+: 266,1676. Found:
m/z 266.1687.
9.29−9.10 (br, 2H), 3.55 (s, 3H, CO2CH3), 3.16−3.12 (m, 2H),
2.78−2.67 (m, 2H), 2.31−2.27 (m, 2H), 1.73−1.69 (m, 2H), 1.46−
1.21 (m, 5H); MS (ES) m/z 172 [M + H]+.
1-Benzyl-4-piperidinepropanol (19).56 To a solution of methyl
4-(piperidin-4-yl)butanoate 1755 (1.60 g, 7.21 mmol) and benzyl
bromide (1.85 g, 10.82 mmol) in 20 mL of CH2Cl2 was added
triethylamine (3.3 mL, 21.65 mmol) while the internal temperature in
the mixture was below 20 °C by cooling with an ice−water bath. The
mixture was stirred at room temperature overnight. After complete
reaction (TLC analysis), the mixture was concentrated and purified by
column chromatography to give methyl 3-(1-benzylpiperidin-4-
yl)propanoate (18) [(1.41 g, 75%): oil; 1H NMR (300 MHz,
CDCl3) δ 7.35−7.23 (m, 5H), 3.66 (s, 3H, CO2CH3), 3.51 (s, 2H,
CH2-Ph), 2.89 (br d, J = 11.5 Hz, 2H), 2.32 (br t, J = 7.5 Hz, 2H, CH2-
CO2CH3), 1.94 (t, J = 10.9 Hz, 2H), 1.66−1.54 (m, 4H), 1.34−1.21
(m, 3H, CH + CH2); MS (EI) m/z (%) 91 (100) [PhCH2]+, 188 (66)
[M − CH2CH2CO2Me]+, 202 (13) [M − CO2Mt]+, 230 (19) [M −
OMe]+, 246 (9) [M − CH3]+, 260 (73) [M − H]+, 261 (50) [M]+],
which was used in the next reaction without further analysis.
N-((5-((1-Benzylpiperidin-4-yl)methoxy)-1-methyl-1H-indol-
2-yl)methyl)-N-methylprop-2-yn-1-amine (3). To a solution of
1-methyl-2-{[ethyl(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol 452
(0.21 g, 0.94 mmol) and 1-benzyl-4-(chloromethyl)piperidine 1027
(0.33 g, 1.51 mmol, 1.5 equiv) in acetonitrile (5 mL), NaH (120 mg, 3
mmol, 3 equiv, 60% mineral oil) was added. The reaction mixture was
stirred at 50 °C for 10 h. After complete reaction (TLC analysis), the
mixture was concentrated, diluted with water, and extracted with
CH2Cl2. The organic phase was washed with brine, dried (MgSO4),
and evaporated. The crude product was purified by flash
chromatography (CH2Cl2/MeOH, 100:1) to give compound 3
(126.3 mg, 32%) as white solid: Rf = 0.24 (CH2Cl2/MeOH, 10:1);
mp 123−5 °C; IR (KBr) ν 3252, 2938, 2913, 1620, 1489, 1466, 1195,
1163, 1029, 1008 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.35−7.24 (m,
5H), 7.18 (d, J = 8.8 Hz, 1H, CH7-indole), 7.04 (d, J = 2.3 Hz, 1H,
CH4-indole), 6.86 (dd, J = 8.8 and 2.3 Hz, 1H, CH6-indole), 6.34 (s,
1H, CH3-indole), 3.85 (d, J = 6.0 Hz, 2H, -CH2O-), 3.73 (s, 3H, N-
CH3), 3.68 (s, 2H, indole-CH2-N), 3.53 (s, 2H, CH2-Ph), 3.31 (d, 2H,
J = 2.2 Hz, CH2-CCH), 2.95 (d, J = 11.4 Hz, 2H), 2.35 (s, 3H, N-
CH3), 2.30 (t, J = 2.2 Hz, CCH), 2.02 (t, J = 16, 2H), 1.91−1.81
(m, 3H), 1.49−1.39 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 153.3
(C5-indole), 138.3 (C1′-Ph), 137.8 (C2-indole), 133.3 (C7a-indole),
129.1 (2 × CH-Ph), 128.1 (2 × CH-Ph), 127.5 (C3a-indole), 126.8
(CH4′-Ph), 111.9 (CH6-indole), 109.5 (CH7-indole), 103.3 (CH4-
indole), 102.0 (CH3-indole), 78.4 (-C), 73.6 (CH2-O), 73.4 (
CH), 63.4 (CH2-Ph), 54.0 (2 × CH2), 63.4 (Ph-CH2), 53.4 (2 ×
CH2), 51.7 (Ind-CH2-N), 44.6 (CH2-C), 41.5 (N-CH3), 35.9 (CH-
piperidine), 29.8 (N-CH3), 29.1 (2 × CH2); MS (EI) m/z (%) 416
(100) [M + H]+, 438 (2) [M +Na]+. 3·2HCl: white powder; mp 230−
3 °C; IR (KBr) ν 3423, 3200, 2933, 2511, 1620, 1486, 1466, 1208
cm−1; 1H NMR (300 MHz, D2O) δ 7.34−7.25 (m, 6H, CH7-indole +
5H-Ph), 7.05 (d, J = 2.2 Hz, 1H, CH4-indole), 6.85 (dd, J = 9.0, 2.2
Hz, 1H, CH6-indole), 6.59 (s, 1H, CH3-indole), 4.47 (s, 2H, CH2),
4.11 (s, 2H, CH2), 3.86 (d, J = 2.4 Hz, 2H, CH2-CCH), 3.79 (t, J =
6.0 Hz, 2H, O-CH2-), 3.59 (s, 3H, indole-CH3), 3.38 (d, J = 12.8 Hz,
2H, CH2), 2.97 (t, J = 2.3 Hz, 1H, CCH), 2.85 (t, J = 12.7 Hz, 2H,
CH2), 2.76 (s, 3H, N-CH3), 1.98 (d, J = 12.8 Hz, 2H, CH2), 1.89−
1.83 (m, 1H, CH), 1.48−1.34 (m, 2H, CH2). Anal. Calcd for
C27H35Cl2N3O: C, 66.39; H, 7.22; Cl, 14.52; N, 8.60. Found: C, 66.21;
H, 7.43; Cl, 14.42; N, 8.63.
To a suspension of LAH (0.176 g, 4.66 mmol, 2 equiv) in dry THF
(10 mL) was added methyl 3-(1-benzylpiperidin-4-yl)propanoate (18)
(0.61 g, 2.33 mmol, 1 equiv) in dry THF (10 mL), at 0 °C. The
mixture was refluxed for 2 h, and after cooling unreacted LiAlH4 was
quenched by careful addition of 10% NaOH solution (20 mL). The
solution was filtered and washed with H2O and EtOAc. The filtrate
was extracted with EtOAc, and the combined organic layers, dried over
Na2SO4, were concentrated to give known compound 1956 (0.535 g,
98%): oil; 1H NMR (300 MHz, CDCl3) δ 7.32−7.20 (m, 5H), 3.60 (t,
J = 6.4 Hz, 2H, CH2O), 3.48 (s, 2H, CH2Ph), 2.87 (br d, J = 11.5 Hz,
2H), 1.92 (br d, J = 11.1 Hz, 2H), 1.68−1.60 (m, 2H), 1.61−1.49 (m,
2H), 1.30−1.15 (m, 5H); MS (EI) m/z (%): 91 (100) [PhCH2]+, 142
(26) [M − CH2Ph]+, 156 (17) [M − Ph]+, 174 (8) [M −
(CH2)3OH]+, 188 (18) [M − (CH2)2OH]+, 202 (14) [M −
CH2OH]+, 232 (37) [M − H]+, 233 (20) [M]+.
1-Benzyl-4-(3-chloropropyl)piperidine (12). To a solution of
1-benzyl-4-piperidinepropanol (19)56 (0.53 g, 2.27 mmol) in CH2Cl2
(5 mL), SOCl2 (0.66 mL, 9.08 mmol, 4 equiv) was added dropwise
with ice cooling. The mixture was refluxed for 3 h and then
evaporated. The residue was rendered alkaline with 10% K2CO3
solution and extracted with CH2Cl2. The organic layer, dried over
Na2SO4, was evaporated under reduced pressure to give compound 12
(0.582 g, 99%): oil; IR 3027, 2923, 2849, 2800, 2756, 1672, 1493,
1452, 1366, 1342, 1263, 738, 698 cm−1; 1H NMR (400 MHz, CDCl3)
δ 7.22−7.36 (m, 5H), 3.51 (t, J = 8.0 Hz, 2H, CH2Cl), 3.51 (s, 2H,
CH2Ph), 2.89 (br d, J = 11.5 Hz, 2H), 1.95 (br t, J = 11.0 Hz, 2H),
1.78 (dt, J = 11.9 and 6.9 Hz, 2H, CH2-CH2Cl), 1.65 (br d, J = 11.4
Hz, 2H), 1.32−1.42 (m, 2H), 1.21−1.30 [m, 3H, CH + CH2-
(CH2)2Cl]; 13C NMR (100 MHz, CDCl3) δ 138.1 (C-Ph), 129.1 (2 ×
CH-Ph), 128.0 (CH-Ph), 126.8 (CH-Ph), 63.3 (CH2-Ph), 53.6
(2CH2-piperidine), 45.2 (CH2Cl), 35.1 (CH), 33.6 [CH2-(CH2)2Cl],
32.1 (2CH2-pieridine), 29.9 (CH2, CH2-CH2Cl); MS (EI) m/z (%) 91
(100) [PhCH2]+, 160 (23) [M − Bn]+, 174 (27) [M − (CH2)3Cl]+,
188 (17) [M − (CH2)2Cl]+, 202 (9) [M − CH2 Cl]+, 216 (88) [M −
Cl]+, 250 (35) [M − 1]+. Anal. Calcd for C15H22ClN: C, 71.55; H,
8.81; N, 5.56. Found: C, 71.84; H, 9.02; N, 5.83.
N-{[5-(2-(1-Benzylpiperidin-4-yl)ethoxy)-1-methyl-1H-indol-
2-yl]methyl}-N-methylprop-2-yn-1-amine (4). To a solution of
1-methyl-2-{[ethyl(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol 1452
(160 mg, 0.7 mmol) and 1-benzyl-4-(2-chloroethyl)piperidine 1153
(0.25 g, 1.05 mmol, 1.5 equiv) in DMF (5 mL), NaH (84 mg, 2.1
mmol, 3 equiv, 60% mineral oil) was added. The reaction mixture was
stirred at room temperature for 3 h. After complete reaction, the
solvent was removed and the crude was diluted with water and
extracted with CH2Cl2. The organic phase was washed with brine,
dried (MgSO4), and evaporated at reduced pressure. The crude
product was purified by flash chromatography (CH2Cl2/MeOH,
100:1) to give compound 4 (0.216 g, 72%) as a white solid: Rf = 0.27
(CH2Cl2/MeOH, 10:1); mp 86−7 °C; IR (KBr) ν 3275, 2941, 2921,
2876, 2807, 2782, 2768, 1619, 1488, 1473, 1289, 1250, 1207, 1161,
1-Benzyl-4-(4-chlorobutyl)piperidine (13). To a solution of 1-
benzyl-4-(4-hydroxybutyl)piperidine (24)57 (0.508 g, 2.05 mmol) in
CH2Cl2 (5 mL), SOCl2 (0.6 mL, 8.214 mmol, 4 equiv) was added
dropwise with ice cooling. The mixture was refluxed for 2 h and then
evaporated. The residue was rendered alkaline with 10% K2CO3
solution and extracted with CH2Cl2. The organic layer, dried over
Na2SO4, was evaporated under reduced pressure to give compound 13
(0.54 g, 99%) as a yellow oil. IR 3062, 3027, 2921, 2847, 2799, 2757,
1493, 1454, 1366, 1341, 1311, 1287, 1126, 1073, 1029, 979, 737, 698
cm−1; 1H NMR (300 MHz, CDCl3) δ 7.34−7.21 (m, 5H), 3.53 (t, J =
6.7 Hz, 2H, CH2Cl), 3.50 (s, 2H, CH2Ph), 2.88 (br d, J = 11.4 Hz,
2H), 1.93 (br t, J = 11.9 Hz, 2H), 1.75 (tt, J = 8 Hz, 2H, CH2-CH2Cl),
1.70−1.59 (m, 2H), 1.49−1.37 (m, 2H), 130−1.20 (m, 5H); 13C
NMR (100 MHz, CDCl3) δ 32.2 (2CH2), 32.7 (CH2, CH2-CH2Cl),
1
1030 cm−1; H NMR (400 MHz, CDCl3) δ 7.34−7.23 (m, 5H), 7.18
(d, J = 8.8 Hz, 1H, CH7-indole), 7.03 (d, J = 2.4 Hz, 1H, CH4-indole),
6.85 (dd, J = 8.8 and 2.4 Hz, 1H, CH6-indole), 6.33 (s, 1H, CH3-
indole), 4.03 (t, J = 6.5 Hz, 2H, O-CH2-), 3.74 (s, 3H, N-CH3), 3.67
(s, 2H, N-CH2), 3.52 (s, 2H, CH2-Ph), 3.31 (d, 2H, J = 2.3 Hz, CH2-
CCH), 2.91 (d, J = 11.6 Hz, 2H), 2.34 (s, 3H, N-CH3), 2.29 (t, J =
2.3 Hz, CCH), 2.0 (t, J = 10.8, 2H), 1.77−1.72 (m, 4H), 1.41−1.31
(m, 2H), 1.62−1.52 (m, CH); 13C NMR (100 MHz, CDCl3) δ 153.5
(C5-indole), 138.4 (C-Ph), 133.6 (C-indole), 137.2 (C-indole), 129.5
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(2 × CH-Ph), 128.3 (2 × CH-Ph), 127.7 (C-indole), 127.2 (CH-Ph),
112.2 (CH6-indole), 109.5 (CH7-indole), 103.5 (CH4-indole), 102.0
(CH3-indole), 78.6 (-C), 73.6 (CH), 66.7 (CH2-O), 63.6 (CH2-
Ph), 53.9 (2 × CH2), 52.0 (CH2-indole), 44.9 (CH2-CCH), 41.8
(N-CH3), 36.2 (CH2), 32.8 (CH2), 32.4 (CH2), 30.1 (N-CH3); MS
(EI) m/z (%) 91 (48) [PhCH2]+, 202 (100), 361 (3) [M −
NCH3CH2CCH)]+, 429 (4) [M]+. Anal. Calcd for C28H35N3O: C,
78.28; H, 8.21; N, 9.78. Found: C, 77.99; H, 8.45; N, 9.79. 4·2HCl:
white powder; mp 221−3 °C; IR (KBr) ν 3424, 3195, 2928, 2561,
(0.5 g, 1.88 mmol, 1.2 equiv) in 8 mL of DMF, NaH (0.1 g, 2.5 mol,
1.6 equiv, 60% mineral oil) was added. The reaction mixture was
stirred at room temperature overnight and then heated at 70 °C for 8
h. Then the mixture was concentrated, diluted with water, and
extracted with CH2Cl2. The organic phase was washed with brine,
dried (MgSO4), and evaporated. The crude product was purified by
flash chromatography (CH2Cl2/MeOH, 50:1 to 30:1, v/v) to give
compound 6 (0.547 g, 76%) as a white solid: Rf = 0.28 (CH2Cl2/
MeOH, 20:1); mp 93−94 °C; IR (KBr) ν 3260, 2937, 2918, 1619,
1
2506, 1619, 1486, 1471, 1210 cm−1; H NMR (300 MHz, D2O) δ
1489, 1472, 1203, 1193, 1160, 1008 cm−1 1H NMR (500 MHz,
;
7.33−7.25 (m, 6H, CH7-indole + 5H-Ph), 7.06 (d, J = 2.4 Hz, 1H,
CH4-indole), 6.84 (dd, J = 9.0, 2.4 Hz, 1H, CH6-indole), 6.58 (s, 1H,
CH3-indole), 4.45 (s, 2H, CH2), 4.08 (s, 2H, CH2), 3.96 (t, J = 6.2
Hz, 2H, O-CH2-), 3.84 (d, J = 2.4 Hz, 2H, CH2-CCH), 3.59 (s, 3H,
indole-CH3), 3.32 (d, J = 12.6 Hz, 2H, CH2), 2.96 (t, J = 2.4 Hz, 1H,
CCH), 2.85−2.75 (m 5H, CH2 + N-CH3), 1.84 (d, J = 13.6 Hz, 2H,
CH2), 1.75−1.63 [m, 1H, CH], 1.61−1.53 (m, 2H, CH2-CH2O-),
1 . 3 2 − 1 . 1 8 ( m , 2 H , C H 2 ) . A n a l . C a l c d f o r
C28H35N3O·2HCl·1/3(H2O): C, 66.13; H, 7.47; Cl, 13.94; N, 8.26.
Found: C, 66.04; H, 7.89; Cl, 13.84; N, 8.59.
CDCl3) δ 7.32−7.23 (m, 5H), 7.17 (d, J = 8.8 Hz, 1H, CH7-indole),
7.03 (d, J = 2.3 Hz, 1H, CH4-indole), 6.85 (dd, J = 8.8 and 2.4 Hz, 1H,
CH6-indole), 6.32 (s, 1H, CH3-indole), 3.98 (t, J = 6.6 Hz, 2H,
-CH2O-), 3.67 (s, 2H, CH2-N), 3.73 (s, 3H, N-CH3), 3.49 (s, 2H,
CH2-Ph), 3.31 (d, J = 2.4 Hz, CH2-CCH, 2H), 2.88 (d, J = 10.5 Hz,
2H, CH2pip), 2.34 (s, 3H, N-CH3), 2.28 (t, J = 2.4 Hz, 1H, CCH),
2.00−1.85 (m, 2H, CH2pip), 1.83−1.73 (m, 2H, CH2-CH2O), 1.66
(br d, J = 9.4 Hz, CH2pip), 1.51−1.45 (m, 2H, CH2-(CH2)2O), 1.34−
1.22 (M, 4H, CH2pip + CH2-(CH2)3O); 13C NMR (125 MHz,
CDCl3) δ 153.5 (C5-indole) 138.5 (C-Ph), 137.0 (C2-indole), 133.3
(C7a-indole), 129.2 (2 × CH2Ph), 128.1 (2 × CHPh), 127.5 (C3a-
indole), 126.8 (CH-Ph), 112.0 (CH6-indole), 109.5 (CH7-indole),
103.4 (CH4-indole), 102.0 (CH3-indole), 78.4 (-C), 73.4 (CH),
68.8 (CH2-O), 63.5 (CH2-Ph), 53.9 (2 × CH2-piperidine), 51.8 (N-
CH2-indole), 44.7 (CH2-CCH), 41.7 (N-CH3), 36.3 (CH2-
(CH2)3O), 35.6 (CH-piperidine), 32.3 (2 × CH2-piperidine), 29.8
(N-CH3), 29.7 (CH2-CH2O), 23.3 [CH2-(CH2)2O]; MS (EI) m/z
(%) 91 (55) [PhCH2]+, 172 (71), 228 (45), 366 (41) [M − Bn]+, 388
[M − NCH3CH2CCH)]+, 418 (8) [M − CH2CCH)]+, 457 (26)
[M]+. Anal. Calcd for C30H39N3O: C, 78.73; H, 8.59; N, 9.18. Found:
C, 78.65; H, 8.71; N, 9.07. 6·2HCl: white powder; mp 197−9 °C; IR
(KBr) ν 3421, 3195, 2928, 2851, 2561, 2509, 1619, 1485, 1472, 1458,
N-{[5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-
indol-2-yl]methyl}-N-methylprop-2-yn-1-amine (5). To a solu-
tion of 1-methyl-2-{[ethyl(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol
452 (0.22 g, 0.963 mmol) and 1-nenzyl-4-(3-chloropropyl)piperidine
12 (0.36 g, 1.44 mmol, 1.5 equiv) in DMF (5 mL), NaH (69.4 mg,
1.73 mmol, 1.8 equiv, 60% /mineral) was added. The reaction mixture
was stirred at room temperature overnight and then heated at 100 °C
for 1 h. After complete reaction (TLC analysis), the mixture was
concentrated, diluted with water, and extracted with CH2Cl2. The
organic phase was washed with brine, dried (MgSO4), and evaporated
at reduced pressure. The crude product was purified by flash
chromatography (CH2Cl2/AcOEt, 10:1 to 5:1, v/v) to give compound
5 (0.268 g, 63%) as a white solid: Rf = 0.28 (CH2Cl2/MeOH, 20:1);
mp 90−91 °C; IR (KBr) ν 3265, 2935, 2908, 2799, 2760, 1619, 1489,
1471, 1395, 1269, 1204, 1190, 1160, 1029 cm−1; 1H NMR (400 MHz,
CDCl3) δ 7.35−7.25 (m, 5H, Ph), 7.19 (d, J = 8.8 Hz, 1H, CH7-
indole), 7.05 (d, J = 2.14 Hz, 1H, CH4-indole), 6.85 (dd, J = 8.8 and
2.3 Hz, 1H, CH6-indole), 6.35 (s, 1H, CH3-indole), 3.98 (t, J = 6.6
Hz, 2H, O-CH2-), 3.75 (s, 3H, indole-CH3), 3.69 (s, 2H, indole-CH2),
3.52 (s, 2H, CH2-Ph), 2.33 (d, J = 2.2 Hz, 2H, CH2-CCH), 2.91 (d,
J = 10.8 Hz, CH2), 2.36 (s, 3H, N-CH3), 2.31 (t, J = 2.0 Hz, CCH),
1.97 (t, J = 12 Hz, 2H, CH2), 1.83 [m, 2H, CH2-(CH2O)], 1.72 (d, J =
9.1 Hz, 2H, CH2), 1.46−1.41 [m, 2H, CH2-(CH2)2O], 1.29−1.31 (m,
3H, CH+CH2); 13C NMR (100 MHz, CDCl3) δ 153.2 (C5-indole),
138.3 (C-Ph), 136.9 (C2-indole), 133.3 (C-indole), 129.2 (2 × CH-
Ph), 128.0 (2 × CH-Ph), 127.4 (C-indole), 126.8 (CH-Ph), 112.0
(CH6-indole), 109.5 (CH7-indole), 103.3 (CH4-indole), 102.0
(CH3-indole), 78.3 (-C), 73.4 (CH), 69.0 (CH2-O), 63.4
(CH2-Ph), 53.8 (2 × CH2), 51.7 (indole-CH2), 44.6 (CH2-C),
41.5 (N-CH3), 35.5 (CH-piperidine), 32.8 [CH2-(CH2)2O], 32.2
(2CH2), 29.8 (indole-N-CH3), 26.7 [CH2-CH2O]; MS (EI) m/z (%)
91 (77) [PhCH2]+, 352 (22) [M − CH2Ph]+, 374 (100) [M −
NCH3CH2CCH)]+, 404 (7) [M − CH2CCH)]+, 428 (5) [M -
CH3]+, 443 (40)[M]+. Anal. Calcd for C29H37N3O: C, 78.51; H, 8.41;
N, 9.47. Found: C, 78.36; H, 8.31; N, 9.23. 5·2HCl: white powder; mp
203−5 °C; IR (KBr) ν 3193, 2937, 2512, 1619, 1486, 1469, 1209
cm−1; 1H NMR (300 MHz, D2O) δ 7.32−7.22 (m, 6H, CH7-indole +
5H-Ph), 7.05 (d, J = 2.2 Hz, 1H, CH4-indole), 6.83 (dd, J = 9.0, 2.3
Hz, 1H, CH6-indole), 6.58 (s, 1H, CH3-indole), 4.45 (s, 2H, CH2),
4.07 (s, 2H, CH2), 3.90 (t, J = 6.1 Hz, 2H, O-CH2-), 3.83 (d, J = 2.2
Hz, 2H, CH2-CCH), 3.59 (s, 3H, indole-CH3), 3.30 (d, J = 12.0 Hz,
2H, CH2), 2.97−2.94 (m, 1H, CCH), 2.81−2.72 (m 5H, CH2 + N-
CH3), 1.84−1.75 (m, 2H, CH2), 1.65−1.55 (m, 2H, CH2), 1.49−1.36
(m, 1H, CH), 1.41−1.46 [m, 2H, CH2-(CH2)2O], 1.29−1.09 (m, 4H).
Anal. Calcd for C29H37N3O·2HCl: C, 67.43; H, 7.61; Cl, 13.73; N,
8.13. Found: C, 67.38; H, 7.81; Cl, 13.13; N, 8.02.
1
1408, 1209 cm−1; H NMR (300 MHz, D2O) δ 7.33−7.24 (m, 6H,
CH7-ind + 5H-Ph), 7.05 (d, J = 2.2 Hz, 1H, CH4-ind), 6.84 (dd, J =
9.0, 2.4 Hz, 1H, CH6-ind), 6.59 (s, 1H, CH3-ind), 4.48 (s, 2H, CH2),
4.05 (s, 2H, CH2), 3.90 (t, J = 6.5 Hz, 2H, O-CH2-), 3.86 (d, J = 2.2
Hz, 2H, CH2-CCH), 3.58 (s, 3H, indole-CH3), 3.28 (d, J = 12.3 Hz,
2H, CH2), 2.98 (t, J = 2.3 Hz, 1H, CCH), 2.77−2.70 (m 5H, CH2 +
N-CH3), 1.76 (d, J = 13.9 Hz, 2H, CH2), 1.61−1.51 (m, 2H, CH2),
1.41−1.23 (m, 3H, CH + CH2), 1.29−1.05 (m, 4H). Anal. Calcd for
C30H39N3O·2HCl: C, 67.91; H, 7.79; Cl, 13.36; N, 7.92. Found: C,
67.54; H, 7.45; Cl, 13.25; N, 8.10;
N-{[5-(2-Bromoethoxy)-1-methyl-1H-indol-2-yl)methyl}-N-
methylprop-2-yn-1-amine (25). A mixture of 1-methyl-2-{[ethyl-
(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol 1452 (0.215 g, 0.942
mmol), 1,2-dibromoethane (1.77 g, 9.42 mmol), and potassium
carbonate (0.65 g, 4.71 mmol) in 2-butanone (8 mL) was reacted at 85
°C for 6 h. The mixture was evaporated in vacuo, and the residue was
partitioned between dichloromethane (10 mL) and water (10 mL).
The organic layer was dried (Na2SO4) and evaporated. The residue
was purified by column chromatography, eluting with 4% methanol in
dichloromethane, affording compound 25 (117.3 mg, 37%): Rf = 0.76
(CH2Cl2/AcOEt, 10:1); mp 75−77 °C; IR 3274, 29712937, 2877,
2800, 1619, 1579, 1488, 1473, 1400, 1267, 1205, 1198, 1159, 1118,
1
1025, 889, 842, 794, 776, 690 cm−1; H NMR (300 MHz, CDCl3) δ
7.2 (d, J = 8.8 Hz, 1H, CH7-indole), 7.07 (d, J = 2.4 Hz, 1H, CH4-
indole), 6.9 (dd, J = 8.8, 2.5 Hz, 1H, CH6-indole), 6.36 (s, 1H, CH3-
indole), 4.33 (t, J = 6.4 Hz, 2H, -CH2-O-), 3.75 (s, 3H, N-CH3), 3.69
(s, 2H, N-CH2), 3.66 (t, J = 6.4 Hz, 2H, -CH2-Br), 3.32 (d, J = 2.4 Hz,
2H, N-CH2-C), 2.36 (s, 3H, N-CH3), 2.31 (t, J = 2.4 Hz, 1H, 
CH); 13C NMR (75 MHz, CDCl3) δ 152.2 (C5-indole), 137.3 (C2-
indole), 133.8 (C7a-indole), 127.4 (C3a-indole), 112.2 (CH6-indole),
109.7 (CH7-indole), 104.4 (CH4-indole), 102.1 (CH3-indole), 78.3
(-C), 73.5 (CH), 69.1 (CH2-O), 51.7 (CH2-N), 44.7 (-CH2-
C), 41.5 (N-CH3), 29.9 (N-CH3), 29.6 (CH2-Br); MS (EI) m/
z(%): 131 (48), 160 (66) [M − ((Br(CH2)2)-NCH3CH2CCH)]+,
267 (100) [M − NCH3CH2CCH)]+, 334 (25)[M]+. Anal. Calcd for
C16H19BrN2O: C, 57.32; H, 5.71; Br, 23.83; N, 8.36. Found: C, 57.50;
H, 5.70; Br, 23.24; N, 8.54.
N-{[5-(4-(1-Benzylpiperidin-4-yl)butoxy)-1-methyl-1H-indol-
2-yl]methyl}-N-methylprop-2-yn-1-amine (6). To a solution of
1-methyl-2-{[ethyl(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol 452
(0.35 g, 1.56 mmol) and 1-benzyl-4-(4-chlorobutyl)piperidine 13
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Journal of Medicinal Chemistry
Article
N-{[5-(2-(4-Benzylpiperidin-1-yl)ethoxy)-1-methyl-1H-indol-
2-yl]methyl}-N-methylprop-2-yn-1-amine (7). 4-Benzylpiperi-
dine (36 μL, 0.2 mmol) was added to a mixture of 25 (34 mg, 0.1
mmol) and potassium carbonate (42 mg, 0.3 mmol) in N,N-
dimethylformamide (1 mL). The reactants were heated at 80 °C
overnight under an atmosphere of argon. The reaction mixture was
poured into water (5 mL) and extracted with dichloromethane (3 × 20
mL). The organic layers were combined, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography,
eluting with 3.3% methanol in dichloromethane, affording compound
extracted into dichloromethane (3 × 20 mL). The organic layers were
combined, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography, eluting with 4% methanol in
dichloromethane, affording compound 8 (89.2 mg, 64%) as white
crystalline solid: Rf = 0.43 (CH2Cl2/MeOH, 10:1); mp 82−3 °C; IR
(KBr) ν 3274, 2923, 1619, 1487, 1469, 1390, 1205, 1133, 1027 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.31−7.25 (m, 2H), 7.21−7.12 (m,
4H), 7.02 (d, J = 2.26 Hz, 1H, CH4-indole), 6.83 (dd, J = 8.83 and
2.43 Hz, 1H, CH6-indole), 6.32 (s, 1H, CH3-indole), 4.03 (t, J = 6.28
Hz, 2H, -CH2O), 3.73 (s, 3H, CH3), 3.67 (s, 2H, CH2-N), 3.30 (d, J =
2.36 Hz, 2H, CH2), 3.02 (d, J = 11.45 Hz, CH2), 2.59 (t, J = 7.37
Hz, CH2), 2.55 (d, J = 6.77 Hz, CH2), 2.34 (s, 3H, CH3), 2.29 (t, J =
2.33 Hz, CCH), 2.08−1.92 (m, 4H, 2 × CH2), 1.66 (d, J = 12.7 Hz,
CH2), 1.55 (m, CH), 1.40 (qd, J = 12.19, 3.24 Hz, CH2); 13C NMR
(100 MHz, CDCl3) δ 153.1 (C5-indole), 140.5 (C-Ph), 137.0 (C2-
indole), 133.3 (C7a-indole), 129.0 (2 × CH-Ph), 128.1 (2 × CH-Ph),
127.4 (C3a-indole), 125.7 (CH-Ph), 111.9 (CH6-indole), 109.5
(CH7-indole), 103.4 (CH4-indole), 102.0 (CH3-indole), 78.3 (
CH), 73.4 (-C), 67.2 (CH2-O), 55.7 (CH2), 53.8 (2 × CH2), 51.7
(CH2), 44.7 (CH2), 43.0 (CH2), 41.5 (N-CH3), 37.7 (CH), 31.7 (2 ×
CH2), 29.8 (CH3), 26.7 (CH2); MS (ES) m/z (%) 188 (99), 444
(100) [M + H]+, 445 (40) [M + 2H]+, 466 (2)[M + Na]+. Anal. Calcd
for C29H37N3O: C, 78.51; H, 8.41; N, 9.47. Found: C, 78.63; H, 8.59;
N, 9.44. 8·2HCl: white powder; mp 216−218 °C; IR (KBr) ν 3196,
2931, 2559, 2509, 1619, 1485, 1472, 1454, 1250, 1211 cm−1; 1H NMR
(300 MHz, D2O) δ 7.27 (d, J = 9.1 Hz, 1H, CH7-indole), 7.20−7.15
(m, 2H), 7.10−7.06 (m, 4H), 6.84 (dd, J = 9.0, 1.5 Hz, 1H, CH6-
indole), 6.60 (s, 1H, CH3-indole), 4.48 (s, 2H, CH2), 3.99 (t, J = 5.4
Hz, 2H, -CH2O), 3.87 (s, 2H, CH2), 3.59 (s, 3H, CH3), 3.41 (d, J =
11.8 Hz, 2H, CH2), 3.16−3.05 (m, 2H), 2.99−2.97 (m,1H, CCH),
2.81−267 (m, 5H, CH2 + N-CH3), 2.43 (d, J = 6.1 Hz, 2H, CH2),
2.06−1.97 (m, 2H, CH2), 1.74−1.70 (m, 3H, CH + CH2), 1.34−1.22
(m, 2H, CH2). Anal. Calcd for C29H37N3O·2HCl·1/2(H2O): C, 66.27;
H, 7.67; Cl, 13.49; N, 8.00. Found: C, 66.02; H, 7.62; Cl, 13.45; N,
8.25.
1
7 (33.5 mg, 77%): white solid; Rf = 0.49 (CH2Cl2/MeOH, 10:1); H
NMR (400 MHz, CDCl3) δ 7.31−7.25 (m, 2H), 7.23−7.14 (m, 4H),
7.06 (d, J = 2.36 Hz, 1H, CH4-indole), 6.88 (dd, J = 8.83 and 2.43 Hz,
1H, CH6-indole), 6.35 (s, 1H, CH3-indole), 4.16 (t, J = 6.06 Hz, 2H,
-CH2-O), 3.74 (s, 3H, CH3), 3.68 (s, 2H, N-CH2), 3.32 (d, J = 2.35
Hz, 2H, CH2), 3.034 (d, J = 11.7 Hz, CH2), 2.83 (t, J = 6.03 Hz, CH2),
2.56 (d, J = 7.016 Hz, CH2), 2.35 (s, 3H, CH3), 2.31 (t, J = 2.37 Hz,
CCH), 2.086 (td, J = 11.77, 2.18 Hz, 1H, CH2), 1.67 (d, J = 12.87
Hz, CH2), 1.56 (m, CH), 1.38 (qd, J = 12.15, 3.79 Hz, CH2); 13C
NMR (100 MHz, CDCl3) δ 152.9 (C5-indole), 140.6 (C-Ph), 137.0
(C2-indole), 133.3 (C7a-indole), 129.0 (2 × CH-Ph), 128.0 (2 × CH-
Ph), 127.4 (C3a-indole), 125.7 (CH-Ph), 111.0 (CH6-indole), 109.5
(CH7-indole), 103.4 (CH4-indole), 102.0 (CH3-indole), 78.3 (
CH), 73.4 (-C), 66.6 (CH2O), 57.6 (CH2), 54.2 (2 × CH2), 51.7
(indole-CH2), 44.65 (CH2), 43.1 (CH2), 41.5 (N-CH3), 37.67 (CH),
32.0 (2 × CH2), 29.8 (CH3); MS (EI) m/z (%) 188 (100), 202 (42),
429 (6)[M]+. 7·2HCl: white powder; mp 218−220 °C; IR (KBr) ν
3421, 3189, 2929, 2498, 1619, 1486, 1208, 1163 cm−1; 1H NMR (300
MHz, D2O) δ 7.28 (d, J = 8.9 Hz, 1H, CH7-indole), 7.20−7.15 (m,
2H), 7.10−7.06 (m, 4H), 6.87 (dd, J = 9.0, 1.9 Hz, 1H, CH6-indole),
6.61 (s, 1H, CH3-indole), 4.49 (s, 2H, CH2), 4.25−4.13 (m, 2H,
-CH2O), 3.87 (d, J = 1.8 Hz, 2H, CH2), 3.60 (s, 3H, CH3), 3.47 (d, J =
12.2 Hz, 2H, CH2), 3.39−3.30 (m, 2H, CH2), 2.98 (t, J = 1.9 Hz, 1H,
CCH), 2.87−2.77 (m, 5H, CH2 + N-CH3), 2.43 (d, J = 6.6 Hz, 2H,
CH2), 1.75−1.71 (m, 3H, CH + CH2), 1.41−1.28 (m, 2H, CH2). Anal.
Calcd for C28H37Cl2N3O·2/3(H2O): C, 65.36; H, 7.51; N, 8.17. Found:
C, 65.08; H, 7.74; N, 8.40.
N-{[5-(3-(4-Benzylpiperazin-1-yl)propoxy)-1-methyl-1H-
indol-2-yl]methyl}-N-methylprop-2-yn-1-amine (9). 1-Benzylpi-
perazine (0.148 g, 0.845 mmol) was added to a mixture of 26 (0.147 g,
0.422 mmol) and potassium carbonate (0.116 g, 0.845 mmol) in N,N-
dimethylformamide (10 mL). The reactants were heated at 70 °C
overnight under an atmosphere of argon. The reaction mixture was
poured into water (50 mL) and extracted into ethyl acetate (3 × 100
mL). The organic layers were combined, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by column
chromatography, eluting with 2% MeOH in CH2Cl2, affording
compound 9 (0.16 g, 85%) as a solid: Rf = 0.43 (CH2Cl2/MeOH,
10:1); mp 103−4 °C; IR (KBr) ν 3138, 2958, 2943, 2806, 2762, 1621,
N-{[5-(3-Bromopropoxy)-1-methyl-1H-indol-2-yl]methyl}-N-
methylprop-2-yn-1-amine (26). A mixture of 1-methyl-2-{[ethyl-
(prop-2-yn-1-yl)amino]ethyl}-1H-indol-5-ol 1452 (21 mg, 0.092
mmol), 1,3-dibromopropane (186 mg, 0.92 mmol), and potassium
carbonate (64 mg, 0.46 mmol) in 2-butanone (1 mL) was reacted at
85 °C for 6 h. The mixture was evaporated, and the residue was
partitioned between dichloromethane (10 mL) and water (10 mL).
The organic layer was dried (Na2SO4) and evaporated in vacuo to give
compound 26 (25.4 mg, 80%): Rf = 0.62 (CH2Cl2/AcOEt, 10:1); mp
1
71−2 °C; IR (KBr) ν 3275, 1488, 1468, 1206, 1026 cm−1; H NMR
1
1492, 1480, 1207, 1159 cm−1; H NMR (400 MHz, CDCl3) δ 7.37−
(400 MHz, CDCl3) δ 7.2 (d, J = 8.9 Hz, 1H, CH7-indole), 7.07 (d, J =
2.4 Hz, 1H, CH4-indole), 6.87 (dd, J = 8.8 and 2.4 Hz, 1H, CH6-
indole), 6.35 (s, 1H, CH3-indole), 4.14 (t, J = 5.8 Hz, 2H, -CH2-OH),
3.75 (s, 3H, N-CH3), 3.69 (s, 2H, ind-CH2-N), 3.65 (t, J = 6.5 Hz, 2H,
-CH2-Br), 3.32 (d, J = 2.4 Hz, 2H, CH2-C), 2.36 (s, 3H, N-CH3),
2.33 [t, J = 5.9 Hz, CH2-(CH2O)], 2.30 (t, J = 2.4 Hz, 1H, CH);
13C NMR (100 MHz, CDCl3) δ 152.8 (C5-indole), 137.1 (C2-indole),
7.25 (m, 5H), 7.19 (d, J = 8.8 Hz, 1H, CH7-indole), 7.07 (d, J = 2.4
Hz, 1H, CH4-indole), 6.88 (dd, J = 8.8 and 2.4 Hz, 1H, CH6-indole),
6.35 (s, 1H, CH3-indole), 4.06 (t, J = 6.4 Hz, 2H, -CH2O), 3.75 (s,
3H, N-CH3), 3.69 (s, 2H, CH2Ph), 3.54 (s, 2H, CH2-N), 3.33 (d, J =
2.3 Hz, 2H, CH2C), 2.53 (m, 8H, 4 × CH2), 2.36 (s, 3H, N-CH3),
2.32 (t, J = 2.3 Hz, 1H, CCH), 2.01 [m, 2H, CH2(CH2O)]; 13C
NMR (100 MHz, CDCl3) δ 153.1 (C5-indole), 138.0 (C-Ph), 136.9
(C2-indole), 133.2 (C7a-indole), 129.1 (2 × CH-Ph), 128.1 (2 × CH-
Ph), 127.4 (C3a-indole), 126.9 (CH-Ph), 111.9 (CH6-indole), 109.5
(CH7-indole), 103.2 (CH4-indole), 101.9 (CH3-indole), 78.3 (-C),
73.4 (CH), 67.1 (CH2-O), 63.0 (CH2-N), 55.33 (CH2), 53.1
(CH2), 53.0 (2 × CH2), 51.7 (CH2), 44.6 (CH2), 41.5 (N-CH3), 29.8
(N-CH3), 26.9 [CH2(CH2O)]; MS (ES) m/z (%) 445 (100) [M +
H]+, 467 (2) [M + Na]+. Anal. Calcd for C28H36N4O: C, 75.64; H,
8.16; N, 12.60. Found: C, 75.39; H, 8.40; N, 12.52. 9·3HCl: white
powder; mp 227−230 °C; IR (KBr) ν 3195, 2953, 2561, 2516, 2442,
133.5 (C7a-indole), 127.2 (C3a-indole), 111.9 (CH6-indole), 109.6
(CH7-indole), 103.6 (CH4-indole), 102.1 (CH3-indole), 78.3 (-C),
73.4 (CH), 66.3 (CH2-O), 51.7 (CH2-N), 44.6 (CH2-C), 41.5
(N-CH3), 32.6 [CH2-(CH2O)], 30.3 (CH2−Br), 29.8 (N-CH3); MS
(EI) m/z (%): 131 (60), 160 (100) [M − ((Br(CH2)3)-
CH3NCH2CCH)]+, 227 (7) [M − (Br(CH2)3)]+, 281 (96)
[CH3NCH2CCH)]+, 348 (21)[M]+. Anal. Calcd for
C17H21BrN2O: C, 58.46; H, 6.06; Br, 22.88; N, 8.02. Found: C,
58.49; H, 6.08; Br, 22.11; N, 8.23.
N-{[5-(3-(4-Benzylpiperidin-1-yl)propoxy)-1-methyl-1H-
indol-2-yl]methyl}-N-methylprop-2-yn-1-amine (8). 4-Benzylpi-
peridine (0.111 mL, 0.632 mmol, 2 equiv) was added to a mixture of
26 (111 mg, 0.316 mmol, 1 equiv) and potassium carbonate (131 mg,
0.648 mmol, 3 equiv) in N,N-dimethylformamide (4 mL). The
reaction mixture was heated at 70 °C for 7 h under an atmosphere of
argon. The reaction mixture was poured into water (5 mL) and
1
1620, 1485, 1472, 1442, 1211 cm−1; H NMR (300 MHz, D2O) δ
7.36−7.31 (m, 5H), 7.27 (d, J = 9.1 Hz, 1H, CH7-indole), 7.05 (d, J =
2.4 Hz, 1H, CH4-indole), 6.84 (dd, J = 9.0, 2.5 Hz, 1H, CH6-indole),
6.61 (s, 1H, CH3-indole), 4.50 (s, 2H, CH2), 4.21 (s, 2H, CH2), 4.00
(t, J = 5.7 Hz, 2H, -CH2O), 3.88 (d, J = 2.5 Hz, 2H, CH2-C), 3.60
(s, 3H, CH3), 3.50−3.35 (m, 4H), 3.33−3.22 (m, 2H), 2.98 (t, J = 2.5
8264
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Journal of Medicinal Chemistry
Article
Hz, 1H, CCH), 2.78 (s, 3H, N-CH3), 2.14−1.98 (m, 2H, CH2).
Anal. Calcd for C28H39Cl3N4O·1/2(H2O): C, 59.73; H, 7.16; N, 9.95.
Found: C, 59.59; H, 7.49; N, 10.20.
Carb 2810TR). Data are the mean ± SEM of at least four different
experiments in triplicate.
Reversibility Studies. To study the nature of the enzymatic
inhibition exerted by 5, we determined the activity of the enzyme in
the presence and in the absence of the inhibitor by two different
methods: after three consecutive washings with buffer and after
different times of preincubation of the enzyme with the inhibitor. For
the first method, enzyme samples were preincubated for 30 min at 37
°C with 6 nM compound 5 for MAO-A and 45 nM for MAO-B.
Samples were then washed and centrifuged at 25000g for 10 min at 4
°C consecutively three times. Finally, total protein was measured and
MAO-A and MAO-B activities were determined as above-described.
For the second method, samples of enzyme and inhibitor 5 at the
indicated concentration were preincubated for 0, 5, 15, and 30 min
before measuring MAO-A and MAO-B activities as above-described.
Progress Curves of Substrate Consumption. To clarify the
behavior of 5 toward MAO-A and MAO-B, the inhibitor was
preincubated for long periods (0−420 min) with MAO-A (10 nM
inhibitor concentration) and MAO-B (100 nM inhibitor concen-
tration). The concentrations of 5 used in this assay were 2 times the
corresponding IC50 value. After the corresponding periods, substrates
were added and MAO activities were determined as above-described.
Data are the mean ± SEM of three independent esperiments in
triplicate.
Kinetic Analysis of MAO-B Inhibition. To obtain estimates of
the mechanism of action of compound 5, reciprocal plots of 1/V
versus 1/[S] were constructed at different concentrations of the
substrate β-phenylethylamine (1−200 μM) by using Fowler and
Tipton’s method.82 The plots were assessed by a weighted least-
squares analysis. Data analysis was performed with GraphPad Prism
3.0 software (GraphPad Software Inc.). Determination of Michaelis
constants gave KM = 6.7 ± 0.3 μM and Vmax = 277.8 ± 6.1 pmol/min.
Slopes of the reciprocal plots were then plotted against the
concentration of 5 (0−10 μM) as previously described79 to evaluate
Ki data.
Inhibitory Capacity on Aβ1−42 Self-Aggregation. The inhib-
ition of Aβ1−42 self-aggregation by compound 5 was studied by using
the thioflavin T-based fluorometric assay previously described by
Bartolini et al.83 with little modifications. Briefly, Aβ1−42 peptide
(Bachem AG, Switzerland) was pretreatted with 1,1,1,3,3,3-hexafloro-
2-propanol (HFIP, Sigma Chemicals) and redissolved in 10 mM
phosphate buffer (PBS, pH 11.2 adjusted with NH4OH). Final Aβ1−42
stock solution concentration was 443 μM. To study the effect of 5 on
fibril formation, experiments were performed by incubating the
peptide (final Aβ concentration of 40 μM) with and without 10 μM
compound 5 (Aβ/inhibitor = 4/1). Blanks containing only the
inhibitor were also prepared. Propidium iodide (PI, Sigma-Aldrich)
was used as reference compound in the experiments at the conditions
described aboved and also at equimolar ratio Aβ/PI. Samples were
diluted to a final volume of 200 μL with 10 mM PBS (pH 7.4), and 35
μM thioflavin T in 50 mM glycine−NaOH buffer (pH 8.5) was added.
Experiments were performed on a Synergy HT microplate
spectrofluorometer (Bio-Tek, U.S.). The fluorescence intensity was
carried out (λexc = 485 nm; λem = 528 nm) every 10 min for 10 h, and
values at plateau (400 min) were averaged after subtracting the
background fluorescence of 35 μM thioflavin T solution. The
fluorescence intensities were compared, and the percent inhibition
due to the presence of the inhibitor was calculated by the following
expression: 100 − [(IFi/IF0) × 100], where IFi and IF0 are the
fluorescence intensities obtained in the presence and absence of
inhibitor, respectively.
Biological Evaluation. Inhibition Experiments of AChE and
BuChE. To assess the inhibitory activity of the compounds toward
AChE (EC 3.1.1.7) or BuChE (EC 3.1.1.8), we followed the
spectrophotometric method of Ellman78 using purified AChE from
Electrophorus electricus (type V-S) or human recombinant (expressed in
the HEK-293 cell line) or BuChE from equine or human serum
(lyophilized powder) (Sigma-Aldrich, Madrid, Spain). The reaction
took place in a final volume of 3 mL of a phosphate-buffered solution
(0.1 M) at pH 8, containing 0.035 U/mL EeAChE, 0.24 U/mL
hrAChE, or 0.05 U/mL BuChE, and 0.35 mM 5,5′-dithiobis-2-
nitrobenzoic acid (DTNB, Sigma-Aldrich, Madrid, Spain). Inhibition
curves were made by preincubating this mixture with at least nine
concentrations of each compound for 10 min. A sample with no
compound was always present to determine the 100% of enzyme
activity. After this preincubation period, acetylthiocholine iodide (0.35
mM) or butyrylthiocholine iodide (0.5 mM) (Sigma-Aldrich, Madrid,
Spain) was added, allowing 15 min more of incubation, where the
DTNB produces the yellow anion 5-thio-2-nitrobenzoic acid along
with the enzymatic degradation of acetylthiocholine iodide or
butyrylthiocholine iodide. Changes in absorbance were detected at
405 nm in a spectrophotometric plate reader (FluoStar OPTIMA,
BMG Labtech). Compounds inhibiting AChE or BuChE activity
would reduce the color generation; thus, IC50 values were calculated as
the concentration of compound that produces 50% AChE activity
inhibition. Data are expressed as the mean ± SEM of at least three
different experiments in quadruplicate.
Kinetic Analysis of AChE Inhibition. To obtain estimates of the
mechanism of action of compound 5, reciprocal plots of 1/V versus 1/
[S] were constructed at different concentrations of the substrate
acetylthiocholine (0.1−1 mM) by using Ellman’s method.78 Experi-
ments were performed in a transparent 48-well plate, each well
containing 350 μL of the DTNB solution in PBS and 1 μL of buffer
(control) or inhibitor solution to give the desired final concentration.
Final volume (1 mL) was reached by adding phosphate buffer solution
(pH 8). Reaction was initiated by adding 45 μL of AChE at 30 °C to
give a final concentration of 0.18 U/mL. Progress curves were
monitored at 412 nm over 1.33 min in a fluorescence plate reader
Fluostar Optima (BMG-Technologies, Germany). Progress curves
were characterized by a linear steady-state turnover of the substrate,
and values of a linear regression were fitted according to Lineweaver−
Burk replots using Origin software. The plots were assessed by a
weighted least-squares analysis. Determination of Michaelis constant
for the substrate ATCh was done at seven different concentrations
(0.1−1 mM) to give KM = 0.29 ± 0.01 mM and Vmax = 2.82 ± 0.06
min−1. Slopes of the reciprocal plots were then plotted against the
concentration of 5 (0−10 μM) as previously described80 to evaluate Ki
data. Data analysis was performed with Origin Pro 7.5 software
(Origin Lab Corp.).
Inhibition Experiments of MAO-A and MAO-B. A purification
of mitochondria from rat liver homogenates was prepared as
previously described81 and used as source for MAO activities. Total
protein was measured by the method of Bradford using bovine-serum
albumin as standard. The inhibitory activity of the compounds toward
MAO-A and MAO-B was determined following the method of Fowler
and Tipton82 using 14C-labeled substrates (Perkinelmer, USA). MAO-
B activity was determined toward 20 μM [14C]phenylethylamine
(PEA) (2.5 mCi/mmol) and MAO-A activity toward 100 μM [14C]-
(5-hydroxy-triptamine) (5-HT) (0.5 mCi/mmol). Inhibition curves
were made by preincubating this mixture with at least nine
concentrations of each compound for 30 min. A sample without
compound was always present to determine the 100% of enzyme
activity. The reaction was carried out at 37 °C in a final volume of 225
μL in 50 mM phosphate buffer (pH 7.2) and stopped by the addition
of 100 μL of 2 M citric acid. Radiolabeled aldehyde product was
extracted into toluene/ethyl acetate (1:1, v/v) containing 0.6% (w/v)
2, 5-diphenyloxazole (PPO) before liquid scintillation counting (Tri-
Inhibition of hAChE-Induced Aβ1−40 Aggregation. Aliquots of
231 μM Aβ1−40 (Bachem AG, Switzerland) lyophilized from 1 mg/mL
HFIP solution were redissolved in 10 mM phosphate buffer (PBS, pH
11.2 adjusted with NH4OH). For co-incubation experiments, aliquots
of human recombinant AChE (Sigma Chemicals) (0.4 μM final
concentration, ratio Aβ/AChE = 10/1) in the presence of 100 μM
compound 5 were added. Blanks containing Aβ, AChE, Aβ plus
compound 5, and AChE plus compound 5 were also prepared. To
quantify the amyloid fibril formation, the thioflavin T fluorescence
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Journal of Medicinal Chemistry
Article
method82 was performed as above-described. The fluorescence
intensities were compared, and the percent inhibition due to the
presence of the inhibitor was calculated by the following expression:
100 − [(IFi/IF0) × 100] where IFi and IF0 are the fluorescence
intensities obtained in the presence and absence of inhibitor,
respectively.
Molecular Modeling. Setup of the Systems. All protein
models were derived from X-ray crystallographic structures taken
from the Protein Data Bank (PDB). AChE models were built up from
the AChE−donepezil complex 1EVE.59 The enzyme was modeled in
its physiological active form with neutral His440 and deprotonated
Glu327, which together with Ser200 forms the catalytic triad. The
standard ionization state at neutral pH was considered for the rest of
the ionizable residues with the exception of Asp392 and Glu443, which
were neutral, and His471, which was protonated, according to previous
studies.84 Three disulfide bridges were defined between Cys residues
66−93, 254−265, 402−521, and histidine residues 398 and 440 were
set up to represent the δ tautomer.85 MAO models were built up using
X-ray structures 2Z5X67 and 2C6586 for isoforms A and B,
respectively. Structural waters were defined as those common to five
different high-resolution X-ray crystallographic structures (PDB entries
2Z5X, 2Z5Y, 2V5Z, 2C70, 2VZ2).
Docking. The binding mode of compound 5 was explored by
means of docking calculations carried out with rDock, which is an
extension of the program RiboDock, using an empirical scoring
function calibrated on the basis of protein−ligand complexes.64,65
Docking computations were performed with a 2-fold purpose: (1) to
explore suitable starting orientations of the inhibitor in the binding site
of AChE, MAO-A, and MAO-B and (2) to examine the docking of
compound 5 for the three main orientations adopted by the indole
ring of Trp279 in the peripheral binding site in Torpedo californica
AChE.61 At this point, it is worth noting that the reliability of rDock
has been assessed by docking a set of known dual binding site IAChEs,
taking advantage of the X-ray crystallographic structures of their
complexes with AChE.60 The docking of 5 in AChE was then explored
using the three structural models of the target AChE differing in the
orientations of Trp279 (PDB entries 1EVE, 1Q83, and 2CKM).
Structural water molecules that mediate relevant interactions between
the benzylpiperidine moiety and the enzyme were retained in the
target models. Similarly, five water molecules found in the binding site
of MAO-A and MAO-B were retained in docking calculations. The
docking volume was defined as the space covered by catalytic, mid-
gorge, and peripheral sites in AChE and by the substrate and entrance
cavities in MAO. Suitable restraints were introduced to position the
benzylpiperidine moiety of 5 in AChE. Each compound was subjected
to 100 docking runs. Whereas the protein was kept rigid, rDock
accounts for the conformational flexibility of the ligand around
rotatable bonds during docking calculations. The output docking
modes were analyzed by visual inspection in conjunction with the
docking scores.
electrostatic contribution was computed using a dielectric constant of
78.4 for the aqueous environment, while a dielectric constant of 1 was
assigned to the interior of the protein. Even though the choice of the
internal dielectric constant is a subject of debate, this value is usually
adopted when calculations are performed for ensembles of snapshots
taken from simulations, whereas higher values are generally used for
calculations of static structures.92,93 The electrostatic potentials were
calculated using a grid spacing of 0.25 Å. Besides the standard atomic
radii implemented in AMBER, calculations were also performed using
a set of optimized radii developed for MM/PBSA computations with
the AMBER force field.66 The nonpolar contribution was calculated
using a linear dependence with the solvent-accessible surface as
implemented in AMBER. Finally, entropy changes upon complexation
were assumed to cancel out in the comparison of the different poses.
Molecular Dynamics. The binding mode of compound 5 was
explored by means of 20 ns molecular dynamics (MD) simulations
performed for their complexes to AChE (using three different models;
see above), MAO-A, and MAO-B. An additional MD simulation was
run for the complex with donepezil and used to calibrate the results of
the simulations performed for AChE complexes. In addition, MD
simulations were also performed for the complexes corresponding to
the covalent adduct formed between 5 and FAD in both MAO-A and
MAO-B. To this end, suitable parameters were developed for the
corresponding imine fragment formed in the chemical reaction
(parameters available upon request to the authors).
The simulation protocol was based on the computational strategy
used in our previous studies,60 which is briefly summarized here. MD
simulations were run using the PMEMD module of AMBER 9 and the
parm99SB parameters for the protein.94 The GAFF force field95,96 was
used to assign parameters to the inhibitor (and to the FAD cofactor in
MAO simulations). The charge distribution of the inhibitor was
further refined based on the electrostatic charges determined from a fit
to the “HF/6-31G(d)” electrostatic potential obtained with Gaussian
0391 using the RESP procedure. Na+ cations were added to neutralize
the negative charge of the system with the XLEAP module of AMBER
9. The system was immersed in an octahedral box of TIP3P97 water
molecules, preserving the crystallographic waters inside the binding
cavity. The final systems contained the protein−ligand complex, Na+
cations, and around 17 000 water molecules, leading to simulations
systems that comprise around 53 000 atoms.
The geometry of the system was minimized in four steps. First, the
position of hydrogen atoms was optimized using 3000 steps of steepest
descent algorithm. Then water molecules were refined through 2000
steps of steepest descent followed by 3000 steps of conjugate gradient.
Next, the ligand, water molecules, and counterions were optimized
with 2000 steps of steepest descent and 4000 steps of conjugate
gradient, and finally the whole system was optimized with 3000 steps
of steepest descent and 7000 steps of conjugate gradient. Thermal-
ization of the system was performed in five steps of 25 ps, increasing
the temperature from 100 to 298 K. Concomitantly, the residues that
define the binding site were restrained during thermalization using a
variable restraining force. Thus, a force constant of 25 kcal mol−1 Å−2
was used in the first stage of the thermalization and was subsequently
decreased by increments of 5 kcal mol−1 Å−2 in the next stages. Then a
series of 20 ns trajectories were run for the two compounds using a
time step of 1 fs. In MAO simulations, an additional restraint force was
used for the backbone of residues 487−492, which define the
transmembrane segment at the C-terminus of the protein. SHAKE was
used for those bonds containing hydrogen atoms, in conjunction with
periodic boundary conditions at constant pressure (1 atm) and
temperature (298 K), particle mesh Ewald for the treatment of long-
range electrostatic interactions, and a cutoff of 11 Å for nonbonded
interactions.
The X-ray structure of the recombinant human BuChE (PDB entry
2PM8)87 was used to explore the binding mode of 5 in this enzyme.
Some graphical manipulation was required, including addition of the
hydrogen atoms according to the parm99SB force field and modeling
of poorly resolved loop between residues Leu478 and Lys486, which
was modeled using the X-ray structure of the BChE−tabun complex
(PDB entry 3DJY).88 Additionally, three disulfide bonds were defined
between residues 94−120, 280−291, and 428−547. Residue Glu469
was modeled in the protonated state, and residue His466 was modeled
as the Nδ-H tautomer. Docking calculations were performed using the
same protocol mentioned above.
MM-PBSA Analysis. The ligand−protein poses were clusterized
and reranked using the MM-PBSA approach in conjunction with the
parmm99 force field of the AMBER (version 9) package.89 The partial
atomic charges of compound 5 were derived using the RESP
protocol90 by fitting to the molecular electrostatic potential calculated
at the HF/6-31G* level with Gaussian 03.91 Calculations were
performed for 100 snapshots taken evenly during the last 5 ns of the
simulations. The internal conformational energy was determined using
the standard formalism and parameters implemented in AMBER. The
ASSOCIATED CONTENT
■
S
* Supporting Information
Representation of structural models derived from docking and
MD simulations, as well as energetic analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Article
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AUTHOR INFORMATION
■
Corresponding Author
*For M.U.: phone, +34-93-5811439; fax, +34-93-5811573; e-
mail, mercedes.unzeta@uab.es. For J.M.-C.: phone, +34-91-
5622900; fax, +34-91-5644853; e-mail, iqoc21@iqog.csic.es.
(11) Riederer, P.; Danielczyk, W.; Grunblatt, E. Monoamine Oxidase-
̈
B Inhibition in Alzheimer’s Disease. Neurotoxicology 2004, 25, 271−
277.
Author Contributions
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⊥These authors have contributed equally to this work.
ACKNOWLEDGMENTS
■
We thank the Ministerio de Ciencia e Innovacio
́
SAF2006-08764-C02-01, SAF2009-07271, and SAF2008-
05595), Generalitat de Catalunya (2009-SGR00298), Comuni-
dad de Madrid (Grant S/SAL-0275-2006), Instituto de Salud
Carlos III [Retic RENEVAS (Grant RD06/0026/1002),
Fundacion CIEN, Miguel Servet Program (Grant CP10/
00531)], and COST Action D34/0003/05 (2005−2010) for
financial support and the Centro de Supercomputació de
Catalunya (CESCA) for computational facilities. A.S. and M.C.
thank CSIC and Instituto de Salud Carlos III for postdoctoral
I3P and “Sara Borrell” contracts, respectively. C.d.l.R. thanks
MICINN for a Juan de la Cierva contract (2006−2008) and
Instituto de Salud Carlos III for a Miguel Servet contract
(2011−2016). J.J.-J. thanks Instituto de Salud Carlos III for
PFIS fellowship FI10/00292. J.M.-C. thanks Prof. A. G. Garcıa
́
(Departamento de Farmacologıa y Terapeu
́
tica, Facultad de
Medicina, Universidad Auto
́
interest and support. This work was realized in the framework
of COST working group: D34/0003/05.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; ACh, acetylcholine; AChE, acetylcho-
linesterase; Aβ, β-amyloid peptide; BuChE, butyrylcholinester-
ase; AChEI, acetylcholinesterase inhibitor; hAChE, human
acetylcholinesterase; Ee, Electrophorus electricus; CAS, catalytic
active site; PAS, peripheral anionic site; MAO, monoamine
oxidase A/B; IMAO, monoamine oxidase inhibitor
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Acetylcholinesterase Inhibitors: Novel Activities of Old Molecules.
Pharmacol. Res. 2004, 50, 441−451.
(24) Munoz-Torrero, D. Acetylcholinesterase Inhibitors as Disease-
̃
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