Design, synthesis, pharmacological evaluation, QSAR analysis, molecular modeling and ADMET of novel donepezil-indolyl hybrids as multipotent cholinesterase/monoamine oxidase inhibitors for the potential treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2013.12.028

The design, synthesis, and pharmacological evaluation of donepezil-indolyl based amines 7-10, amides 12-16, and carboxylic acid derivatives 5 and 11, as multipotent ASS234 analogs, able to inhibit simultaneously cholinesterase (ChE) and monoamine oxidase (MAO) enzymes for the potential treatment of Alzheimer's disease (AD), is reported. Theoretical studies using 3D-Quantitative Structure-Activity Relationship (3D-QSAR) was used to define 3D-pharmacophores for inhibition of MAO A/B, AChE, and BuChE enzymes. We found that, in general, and for the same substituent, amines are more potent ChE inhibitors (see compounds 12, 13versus7 and 8) or equipotent (see compounds 14, 15versus9 and 10) than the corresponding amides, showing a clear EeAChE inhibition selectivity. For the MAO inhibition, amides were not active, and among the amines, compound 14 was totally MAO A selective, while amines 15 and 16 were quite MAO A selective. Carboxylic acid derivatives 5 and 11 showed a multipotent moderate selective profile as EeACE and MAO A inhibitors. Propargylamine 15 [N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl) prop-2-yn-1-amine] resulted in the most potent hMAO A (IC₅₀ = 5.5 ± 1.4 nM) and moderately potent hMAO B (IC₅₀ = 150 ± 31 nM), EeAChE (IC₅₀ = 190 ± 10 nM), and eqBuChE (IC ₅₀ = 830 ± 160 nM) inhibitor. However, the analogous N-allyl and the N-morpholine derivatives 16 and 14 deserve also attention as they show an attractive multipotent profile. To sum up, donepezil-indolyl hybrid 15 is a promising drug for further development for the potential prevention and treatment of AD.

Full text of DOI:10.1016/j.ejmech.2013.12.028

European Journal of Medicinal Chemistry 75 (2014) 82e95
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, pharmacological evaluation, QSAR analysis,
molecular modeling and ADMET of novel donepezileindolyl hybrids
as multipotent cholinesterase/monoamine oxidase inhibitors for the
potential treatment of Alzheimer’s disease
,
,
Oscar M. Bautista-Aguilera a 1, Gerard Esteban b 1, Irene Bolea b, Katarina Nikolic c,
Danica Agbaba c, Ignacio Moraleda d, Isabel Iriepa d, Abdelouahid Samadi a, Elena Soriano e,
Mercedes Unzeta b, José Marco-Contelles a
,
*
a Laboratorio de Química Médica (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
b Departament de Bioquímica i Biología Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
c Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
d Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.5, 28817 Alcalá de Henares, Spain
e SEPCO, (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and pharmacological evaluation of donepezileindolyl based amines 7e10, amides
12e16, and carboxylic acid derivatives 5 and 11, as multipotent ASS234 analogs, able to inhibit simul-
taneously cholinesterase (ChE) and monoamine oxidase (MAO) enzymes for the potential treatment of
Alzheimer’s disease (AD), is reported. Theoretical studies using 3D-Quantitative StructureeActivity
Relationship (3D-QSAR) was used to define 3D-pharmacophores for inhibition of MAO A/B, AChE, and
BuChE enzymes. We found that, in general, and for the same substituent, amines are more potent ChE
inhibitors (see compounds 12, 13 versus 7 and 8) or equipotent (see compounds 14, 15 versus 9 and 10)
than the corresponding amides, showing a clear EeAChE inhibition selectivity. For the MAO inhibition,
amides were not active, and among the amines, compound 14 was totally MAO A selective, while amines
15 and 16 were quite MAO A selective. Carboxylic acid derivatives 5 and 11 showed a multipotent
Received 22 October 2013
Received in revised form
11 December 2013
Accepted 22 December 2013
Available online 27 January 2014
Keywords:
Donepezil
Donepezileindolyl hybrids
EeAChE
moderate selective profile as EeACE and MAO
A inhibitors. Propargylamine 15 [N-((5-(3-(1-
eqBuChE
hMAO A
hMAO B
Inhibitors
3D-QSAR
Molecular modeling
ADMET
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)prop-2-yn-1-amine] resulted in the
most potent hMAO A (IC50 ¼ 5.5 ꢀ 1.4 nM) and moderately potent hMAO B (IC50 ¼ 150 ꢀ 31 nM), EeAChE
(IC50 ¼ 190 ꢀ 10 nM), and eqBuChE (IC50 ¼ 830 ꢀ 160 nM) inhibitor. However, the analogous N-allyl and
the N-morpholine derivatives 16 and 14 deserve also attention as they show an attractive multipotent
profile. To sum up, donepezileindolyl hybrid 15 is a promising drug for further development for the
potential prevention and treatment of AD.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Acetylcholinesterase inhibitors, such as donepezil, galanth-
amine, and rivastigmine, increase the ACh concentration in the
Alzheimer’s disease (AD) is a neurodegenerative disorder char-
acterized by dementia and other cognitive impairments [1]. From
synaptic cleft, but with limited success and efficiency in the current
clinical therapy for AD [5e7].
the biochemical point of view, amyloid-
b
(A
b
)
and hyper-
Similarly, some symptoms of AD are due to alterations in the
dopaminergic, serotoninergic and monoaminergic neurotrans-
mitter systems [8e10]. In particular, monoamine oxidases (MAO) A
and B are the enzymes that catalyze the oxidative deamination of
biogenic amines [11], rendering the corresponding aldehyde,
ammonia and hydrogen peroxide as metabolic products. Thus, MAO
inhibitors (MAOI) might increase amine neurotransmission and
reduce reactive oxygen species affording potential valuable effect
phosphorylated -protein aggregates [2], oxidative stress [3] and
s
low levels of acetylcholine (ACh) [4] play, among other, key roles in
the in the progress and development of AD.
* Corresponding author.
E-mail address: iqoc21@iqog.csic.es (J. Marco-Contelles).
These authors have equally contributed to this work.
1
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.028

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
83
for the treatment of AD [11,12] by decreasing the neuronal damage
[13,14]. Recent studies have shown that the propargylamine group
present on MAOIs, such as rasagiline and ladostigil, is the respon-
sible for the neuroprotective, anti-apoptotic [15e17], and Ab ag-
gregation inhibition power of these compounds [18].
Unfortunately, and despite the recent advances reported, no
efficient therapy has been implemented for the treatment of AD
patients. This is possibly due to the multifactorial and complex
nature of AD, which makes inadequate the use of magic bullets
targeted to a single receptor or enzymatic system for the efficient
treatment of the disease [7,15].
However, it is now widely accepted that a more effective ther-
apy would result from the use of multipotent compounds able to
intervene in the different pathological events underlying the eti-
ology of AD [19,20].
In the last years our laboratory has been particularly active in
this area, and very recently we have described a number of multi-
potent MAO/ChE inhibitors [21,22]. These drugs, exemplified by
compound ASS234 {N-[5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-
methyl-1H-indol-2-yl]methyl]-N-methylprop-2-yn-1-amine]} [22]
(Chart 1), are hybrids of donepezil, an AChE inhibitor, and
PF9601N [N-((5-(benzyloxy)-1H-indol-2-yl)methyl)prop-2-yn-1-
amine] (Chart 1) [23], a potent and selective MAO B inhibitor [23].
PF9601N is also an effective neuroprotective agent in several in vivo
models of Parkinson’s disease, attenuates the MPTP-induced striatal
dopamine depletion in young-adult and old-adult C57/BL mice,
reduces the loss of tyrosine hydroxylase (TH)-positive neurons after
nigrostriatal injection of 6-hydroxydopamine in rats [24], and en-
Chart 2. New multipotent ChE þ MAO inhibitors 3e16 described in this work.
primary alcohol in 11. Precedents that support this selection are
known in the recent literature [27]. Similarly, ASS234 analogs of
type II have been considered, resulting in the secondary amines
12e16 (Chart 2) when substituting the N(Me)propargylamine motif
by N(H)benzyl, N(H)phenyl, morpholino, N(H)propargyl and N(H)
allyl derivatives, respectively. Note that the use of the morpholino
motif is not casual, as it is found in moclobemide, a well-known
selective MAO A reversible, non-covalent inhibitor [28]. Finally,
simple O-methyl ethers 3 and 4 (Chart 2) have been prepared in
order to evaluate the pharmacological effect of the donepezil-like
motif present in related compounds 7 and 12 (Chart 2).
hances the duration of L-3,4-dihydroxyphenylalanine (L-DOPA)-
In this work we have also applied and described the 3D-QSAR
approach in order to design potent inhibitors of ChEs and MAO,
with optimal selectivity profile. The main aim of the 3D-QSAR study
was to define specific molecular determinants for MAO A/B, AChE,
and BuChE inhibition and to create QSAR models for evaluation of
MAO A/B, AChE, and BuChE activity of the designed multipotent
inhibitors.
induced contralateral turning in 6-hydroxydopaminelesioned rats
[25].
Compound ASS234 inhibits both MAO A/B, AChE and BuChE
enzymes. In addition, recent studies have shown that hybrid
ASS234 retains the anti-apoptotic and antioxidant properties
observed by the parent compound PF9601N, and possesses a
favorable bloodebrain barrier permeability [26]. These findings
suggest that ASS234 is a new promising multitarget drug candidate
for the potential treatment of AD.
2. Results and discussion
Now, in this context, we have designed, synthesized and sub-
mitted to pharmacological evaluation the new multipotent
ChE þ MAO inhibitors of type I and II (Chart 1). The structure of
inhibitors of type I, such as 5e10 (Chart 2), is based on donepezil
(1) and ASS234 (2), by fixing the N-benzylpiperidine motif linked
by a three methylene carbon chain to the oxygen at C5 in the indole
nucleus, while the methylene group at C2 becomes a carbonyl
group, resulting in ester 5, carboxylic acid 6, amides 7e10, or a
2.1. Chemistry
The synthesis of the target molecules 3e16 has been carried out
as shown in Schemes 1e3 (see Experimental part).
The synthesis of N-benzyl-5-methoxy-1-methyl-1H-indole-2-
carboxamide (3) is shown in Scheme 1. Thus, the reaction of ethyl
5-methoxy-1-methyl-1H-indole-2-carboxylate (17) [29] with so-
dium hydroxide in ethanol, at reflux, gave the corresponding car-
boxylic acid 18 [30], whose reaction with benzylamine, in the
presence of DDC/HOBT, to afford amide 3, followed by reduction with
lithium aluminum hydride (LiAlH4), provided amine 4 in 79% yield.
Reaction of 1-benzyl-4-(3-chloropropyl)piperidine (19) [22] and
ethyl 5-hydroxy-1-methyl-1H-indole-2-carboxylate (20) [31], in
the presence of potassium carbonate, in DMF, gave product 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carboxylate
(5) in 63% yield, which after hydrolysis with lithium hydroxide,
yielded
5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-
indole-2-carboxylic acid (6), whose reaction with phosphorus
pentachloride provided acid chloride (21) (Scheme 2). This inter-
mediate, without further purification or isolation, was reacted with
the selected amines, such as benzylamine, aniline, morpholine and
propargylamine, under the usual conditions, to give amides 7e10 in
good yields (Scheme 2).
Next, {5-[3-(1-benzylpiperidin-4-yl) propoxy]-1-methyl-1H-
indol-2-yl} methanol (11) was obtained by reduction of the ester 5
with LiAlH4 in dry THF (Scheme 3).
Chart 1. General structure of donepezil, ASS234, PF9601N, and the novel MAOI/ChEI
hybrids I and II, described in this work.

84
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
All the amines 12e16 were prepared from the corresponding
amides 7e10 by reaction with LiAlH4 (Scheme 2).
range that ASS234 (2). Finally, it was very interesting to find that
the alcohol 11, ester 5 and carboxylic acid 6 are selective and potent
Interestingly, in the case of the reduction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-N-(prop-2-yn-1-yl)-1H-
indole-2-carboxamide (10), besides the expected amine 15 (51%),
the N-allylamine 16 (29%) was also isolated. The structure of this
product was easily deduced by inspection of its analytic and
AChEI, in the 0.24e0.40 mM range.
Regarding the MAO inhibition (see Table 1), unfortunately, most
of the indoles proved inactive. This is the case of amides 3, 4, 7e10,
or amines 12 and 13, while only amines bearing the morpholino
(14), the N-propagylamino (15) or the N-allylamino (16) groups
were potent and selective MAO A inhibitors. Note also that again
the alcohol 11 and the ester 5, but not the acid 6, were quite potent
and selective MAO A inhibitors. Particularly, and not surprisingly,
indole 15 is a potent MAO A inhibitor [(IC50 ¼ 5.5 ꢀ 1.4 nM)], in the
same range that ASS234 (2), but 3.5-fold less potent than ASS234
(2) for the inhibition of hMAO B.
To sum up, and looking for new multipotent ChE/MAO inhibitors
for AD, and concerning our effort to improve the pharmacological
results with ASS234 (2) [22], we can conclude that donepezil-like
indole derivative 15, bearing the N(1)Me/N(H)propargylamine
structural motif, is a very potent and selective EeAChE and hMAO A
inhibitor showing also a non-negligible eqBuChE and hMAO B ac-
tivities, comparing very favorably with hybrid ASS234 (2) regarding
the EeAChE and MAO A inhibition, but showing a poorer eqBuChE
and MAO B inhibition profile than ASS234 (2).
spectroscopic data. In particular, the presence of signals at
d 5.91
(m, 1H, NCH2CH]CH2), 5.21 (dd, J ¼ 17.1 and 1.5 Hz, 1H, NCH2CH]
CH2), 5.12 (dd, J ¼ 10.3 and 1.5 Hz,1H, NCH2CH]CH2), and at
d 136.6
(NHCH]CH2) and 116.2 (NHCH]CH2), in the 1H and 13C NMR
spectra, respectively, corresponded to a double bond in an N-
allylamine, obtained as the result of further reduction of the acet-
ylenic bond in the corresponding propargylamine.
All new compounds showed analytical and spectroscopic data
were in good agreement with their structure, and data reported in
the literature for similar compounds (see Experimental part).
2.2. Pharmacological evaluation
The in vitro activity of these new indoles 3e16 against EeAChE
and eqBuChE was determined using Ellman’s method [32].
In order to test their multipotent profile, compounds 3e16 have
also been evaluated as human MAO A/B inhibitors following a
fluorometric method [33], giving the IC50 values shown in Table 1,
with donepezil (1) and ASS234 (2) as reference compounds.
Regarding the ChEs inhibition, the active indole derivatives here
analyzed are from moderate to potent, in the low micromolar
range, EeAChE inhibitors. For the selectivity, EeAChE is in general
more strongly inhibited than eqBuChE. Compounds 5, 6, 9e11 are
EeAChE selective, while indoles 12e16 show a remarkable eqBuChE
inhibition power. Note also that amides 3, 7 and 8 do not inhibit the
ChEIs. The most potent and selective AChE inhibitor was amine 13
2.3. QSAR Analysis of novel donepezileindolyl hybrids
In Supplementary material, a 3D-QSAR study has been carried
out on these new indoles to explain and predict the binding on the
active sites of the enzymes based on the pharmacological activities
observed on these derivatives.
The created 3D-pharmacophore models are further applied in
order to design novel multipotent inhibitors and for the selection of
new promising ligands (Chart 2). The designed donepezileindolyl
hybrids have been synthesized and examined for MAO/ChE inhib-
iting activity (Tables 2 and 3). As shown, the agreement between
predicted and measured MAO/ChE inhibiting activity for the novel
ligands confirmed the predicting capacity of the formed 3D-QSAR
models. Therefore the 3D-QSAR models could be further used for
designing and the evaluation of novel donepezileindolyl hybrids.
(IC50 ¼ 0.084 ꢀ 0.016
mM), bearing a N-phenyl residue in the
aminomethyl chain at C2. Compared to donepezil (1), indole 13
results 6.6-fold less active for the AChE inhibition and almost
equipotent for BuChE. We found that, in general, and for the same
substituent, amines are more potent (see compounds 12,13 versus 7
and 8) or equipotent (see compounds 14, 15 versus 9 and 10) than
the corresponding amides. While this is not surprising taking into
account that amide 3 is not a donepezil-like derivative, the results
observed for amides 7 and 8 allowed us to conclude that the
presence of a N-benzyl or N-phenyl substituent at the aminomethyl
chain at C2 is detrimental for the ChE activity.
2.4. Molecular modeling
2.4.1. Molecular modeling of compound 15. Inhibition of AChE and
BuChE
To shed light onto the putative orientation of the cholinesterase
inhibitor 15 in both cholinesterases, molecular modeling studies
were carried out using the Autodock Vina software [34]. A structure
of EeAChE (PDB: 1C2B) was used for the in silico studies and
exploited in the in vitro biochemical assays presented above. The
binding site for docking was designed such that the entire receptor
molecule was included within the selection grid. Eight side chains,
Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75, Trp86 and Tyr341 are
allowed to change their conformations at the same time as the
ligand that is being docked.
Comparing compounds 3 and 4, the effect of the amide
(“CONHR”) versus the corresponding amine (“CH2NHR”) is clear and
positive, rendering now amine 4 a moderately active and selective
AChEI. Note also that the moclobemide-like compound 14 is a
potent and moderately selective AChE inhibitor, while the
moclobemide-like amide 9 is totally AChE selective. Compared with
ASS234 (2), the N-propargylated analogs 10 and 15 are 2.6 and 1.8-
fold, respectively, more potent for the inhibition of AChE. However,
and regarding the BuChE inhibition, amide 10 is inactive, but
compound 15 is 1.8-fold less active than ASS234 (2). The observed
values for N-propargylamine 15 and N-allylamine 16 deserve also
some comment, as compound 16 shows a similar EeAChE inhibition
power, but a slightly better eqBuChE inhibitory profile, in the same
The binding mode for 15 (ꢁ11.9 kcal/mol) confirmed that the
indole fragment was bound to near the bottom of the gorge (Fig. 1).
The propargylamine scaffold of inhibitor 15 exerted strong parallel
pep stacking to Trp86. In the middle of the gorge, the aliphatic
Scheme 1. Reagents and conditions: (a) NaOH 10 N, EtOH, reflux (93%); (b) HOBT, DIPEA, DCC, benzylamine, DMF; (c) LiAIH4, THF.

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
85
Scheme 2. Reagents and conditions: (a) K2CO3, dry DMF, reflux; (b) LiOH, EtOH, reflux; (c) PCI5, CH2CI2; (d) RNH2 (or morpholine) TEA, CH2CI2; (e) LiAIH4, dry THF.
alkyl chain of compound 15 was surrounded with the phenyl rings
of Tyr124, Tyr341 and Phe338 providing 15eEeAChE complex,
stabilization and constriction. At the rim of the gorge within pe-
ripheral anionic site, the phenyl ring is stacked between Trp286 and
Tyr72 (Fig. 1).
Analysis of the intermolecular interactions indicated key resi-
dues responsible for ligand binding. The propargylamine group is
likely to be an important feature for these derivatives to exhibit
both AChE- and BuChE-inhibitory activities.
It is significant to note that the linear conformation allows
compound 15 to span both the catalytic active site (CAS) and PAS,
contributing to its superior binding toward AChE.
The docking study of 15 within the active site of eqBuChE was
also carried out. In the absence of X-ray structure of eqBuChE, a
homology model was used. SWISS-MODEL [35e37], a fully auto-
mated protein structure homology-modeling server was used to
design the receptor. A putative three-dimensional structure of
eqBuChE has been created based on the crystal structure of hBuChE
(pdb: 2PM8) as these two enzymes exhibited 89% sequence iden-
tity. In order to simulate the binding of compound 15 to eqBuChE,
docking experiments were performed as blind dockings following
the same computational protocol used for EeAChE. As depicted in
Fig. 2, compound 15 was well accommodated inside the gorge
active site. The orientation of 15 in the eqBuChE active site was
different from what observed in EeAChE, showing a binding mode
where the ligand adopted an U-shaped conformation. The indole
ring faced to Ser198 and Hys438 and the nitrogen atom of the
propargylamine moiety is bridged to His438 via hydrogen bonding.
2.4.2. Molecular modeling of compound 15. Inhibition of MAO A
and MAO B
Compound 15 was docked into the active site of both MAO A and
MAO B isoforms using the program Autodock Vina [34] in order to
structurally understand the interactions with these biotargets
together with the inhibitory profiles. Since inhibition data were
determined on human MAOs, docking simulations were run on the
human model of the MAO A and MAO B. The 3D structures for
hMAOs were retrieved from the PDB (PDB ID: 2Z5X for hMAO A and
PDB ID: 2V5Z for hMAO B).
Results from several studies have shown that it must be the
neutral amine that reaches the active site of MAO A and MAO B to
allow the chemistry [38e41]. The docking simulations were done
with compound 15 as neutral specie despite of at physiological pH,
most of the piperidine rings would be in the protonated, positively
charged form.
Besides,
pep interaction can be observed within propargylamine
moiety and Trp82. The phenyl group is in T-shaped orientation with
respect to Trp231.
Scheme 3. Synthesis of compound 11.

86
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
Table 1
Inhibition of AChE from Electrophorus electricus (EeAChE), equine serum butyrylcholinesterase (eqBuChE) and human monoamine oxidase (hMAO A/B) by donepezil (1),
ASS234 (2) and compounds 3e16.
Compound
R
IC50
(
m
M)a
Selectivity
IC50
(m
M)a
Selectivity
EeAChE
eqBuChE
eqBuCh/EeAChE
hMAO A
hMAO B
hMAO B/hMAO A
Donepezil (1)
ASS234 (2)
3
4
5
6
0.013 ꢀ 0.001
0.35 ꢀ 0.01
>100
15.2 ꢀ 2.1
0.27 ꢀ 0.02
0.40 ꢀ 0.04
0.84 ꢀ 0.05
0.46 ꢀ 0.06
>100
65
1.3
>1
1.5
>370
>250
850 ꢀ 13
0.0042 ꢀ 0.0005
>100
15 ꢀ 2.2
0.039 ꢀ 0.004
>100
>100
>100
0.017
9.2
>1
>1
>65
>1
22.9 ꢀ 5.1
>100
OEt
OH
>100
1.53 ꢀ 0.21
>100
>100
>100
7
>100
>100
>1
>100
>100
>100
>100
>1
8
>100
>100
>1
>1
9
0.27 ꢀ 0.02
>100
>370
>100
>100
>1
10
11
12
0.13 ꢀ 0.02
0.24 ꢀ 0.03
0.14 ꢀ 0.02
>100
>770
>416
2
>100
>100
>100
>100
>1
>23
>1
OH
>100
4.26 ꢀ 0.56
>100
0.29 ꢀ 0.07
13
0.084 ꢀ 0.016
0.10 ꢀ 0.01
0.85 ꢀ 0.25
10
>100
>100
>100
>1
14
15
0.79 ꢀ 0.077
7.9
4.40 ꢀ 0.84
>22.7
0.19 0.01
0.83 0.16
4.4
0.0055 0.0014
0.15 0.031
27.3
16
0.14 ꢀ 0.01
0.46 ꢀ 0.08
3.3
0.98 ꢀ 0.22
15.3 ꢀ 2.5
15.6
a
Values are expressed as mean ꢀ standard error of the mean of at least three different experiments in quadruplicate.
Table 3
Experimental and QSAR activities of novel EeAChE and eqBuChE inhibitors.
Table 2
Experimental and QSAR activities of novel MAO A and MAO B inhibitors.
Compound
R
pIC50
Pred pIC50 pIC50
Pred pIC50
Compound
R
pIC50
Pred-pIC50 pIC50
Pred-pIC50
(EeAChE) (EeAChE) (eqBuChE) (eqBuChE)
(hMAO A) (MAO A)
(hMAO B) (MAO B)
Donepezil (1)
Donepezil (1)
4
4.818
6.854
6.252
7.695
4.640
6.538
4.876
6.188
4
4.000
4.000
4.912
7.806
4.000
4.000
6.275
6.099
12
13
12
13
7.076
6.399
6.071
6.051
4.000
6.875
4.000
6.127
15
16
6.721
6.854
7.524
8.150
6.081
6.337
6.442
6.093
15
16
8.260
6.009
8.146
8.590
6.824
4.815
6.000
6.219

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
87
the “entrance cavity”, while the indole ring is well accommodated
by the catalytic cleft, leaving the propargylamine substituent facing
the FAD cofactor. Glu215 contributes to the stabilization of complex,
acting as a hydrogen bond acceptor with the amino group of the
propargylamine moiety also involving a water molecule. Further-
more, the highly hydrophobic environment of the “entrance cavity”
contributes to the stabilization of the ligand, as the N-benzylpi-
peridine moiety undergoes stable hydrophobic interactions with
Val93, Leu97 and Ala111.
Slightly different binding features were revealed in analysis of
the results obtained for the Mode I relative Mode II. In Mode II
(ꢁ9.3 kcal/mol, in yellow), compound 15 is turned by 180ꢂ with
respect to the position adopted in Mode I. The N-benzyl moiety
binds the “entrance cavity” of the receptor with the phenyl ring
directed toward Thr205, while the propargylamine motif is stably
linked to the substrate cleft. On one hand, the propargyl group
forms favorable pep stacking interactions with Tyr407, Tyr444 and
FAD cofactor, while the amino group of the propargylamine moiety
interacts with the carbonyl oxygen atom of the Gln215 via a
hydrogen bond. A hydrogen bond interaction occurs between the
ether oxygen of the compound 15 and the thiol group of Cys323.
It should be noticed that the propargyl group present in com-
pound 15, is faced to the FAD cofactor. By analogy with the prop-
argyl containing MAO A inhibitors, such as clorgyline, able to
inactivate the enzyme by covalent interactions with the N5 of FAD
isoalloxazine [43], compound 15 could also establish such covalent
interactions via their propargyl group, which can be considered the
irreversible-enzyme inactivating moiety.
Fig. 1. Binding mode of inhibitor 15 at the active site of EeAChE. Compound 15 is
illustrated in blue. Ligands are rendered as balls and sticks and the side chains con-
formations of the mobile residues are illustrated in the same color light as the ligand.
Different subsites of the active site were colored: catalytic triad (CT) in green, oxyanion
hole (OH) in pink, anionic sub-site (AS) in orange, except Trp86, acyl binding pocket
(ABP) in yellow, and peripheral anionic subsite (PAS) in blue. Black dashed lines are
drawn among atoms involved in hydrogen bond interactions. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
As reported in our previous paper [42], a number of six struc-
tural water molecules were explicitly considered in the docking
simulations. These water molecules are labeled as w72, w193, w11,
w23, w15, and w53 in accordance with the numbering reported for
the hMAO B crystallographic structure (PDB ID: 1S3E) and they are
located near the FAD cofactor.
Analysis of the binding modes revealed that compound 15 could
bind to hMAO A in two modes (Mode I and Mode II) as shown in
Fig. 3. In Mode I (ꢁ9.4 kcal/mol, in red), the benzyl moiety occupies
We also docked compound 15 into hMAO B to study the
different behavior against both isoenzymes, MAO A and MAO B.
Blind docking studies were done and the six structural water
molecules selected for hMAO A were also included in the study.
Regarding MAO B binding modes, our findings revealed that
unique pattern recognition is present (ꢁ8.8 kcal/mol). This inhibi-
tor also crosses both cavities, presenting the piperidine nucleus
located between the “entrance” and “catalytic” cavities, separated
by the residues Ile199 and Tyr326 (Fig. 4). The substituted-indole
ring is oriented toward the bottom of the substrate cavity, inter-
acting with the FAD cofactor through pep interactions. In addition,
the predicted orientation of the substituted-indole moiety allowed
the interaction of the NH hydrogen of the propargylamine moiety
with a water molecule, thereby preventing a hydrogen bond
interaction with the enzyme. Finally, the phenyl ring is oriented to
an entrance cavity, a hydrophobic sub-pocket, which is defined by
Leu 88, Pro102 and Ile316.
The study confirmed the selectivity of compound 15 for MAO A.
Selectivity is likely due to the orientation of the propargylamine
and phenyl moieties of 15 in MAO A and in MAO B. The pose ob-
tained in hMAO B for compound 15 showed a preference for the
ligand to be placed in a deeper region (see Fig. 4b), resulting in an
energetically less favorable complex. Compound 15 established
more interactions with the MAO A active site compared to the MAO
B active site which may indicate the 15 interacts more tightly with
MAO A.
2.5. ADMET analysis
A variety of key ADMET (Absorption, Distribution, Metabolism,
Excretion and Toxicity) properties has been calculated, with special
emphasis on the requirements of the central nervous system (CNS).
These requirements for CNS penetration by drugs are reasonably
well understood and accordingly many theoretical models have
been developed, although additional work still needs to be done on
the effect and optimal range of molecular structure on penetration.
The drugs used for neurological disorder treatment, such as AD, are
Fig. 2. Complex of compound 15 and eqBuChE homology built 3D-model. Compound
15 is illustrated in blue sticks. Black dashed lines are drawn among atoms involved in
hydrogen bond interactions. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

88
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
Fig. 3. Docking pose of inhibitor 15 into hMAO A. (a) Crystal structure of hMAO A represented in ribbon diagram. Binding modes I and II are represented as red and yellow sticks,
respectively. (b) Amino acid residues of the binding site are color-coded. The flavin adenine dinucleotide cofactor (FAD) and the six water molecules are represented as an integral
part of the MAO A structure model and are rendered as orange sticks and red balls, respectively. Green dashed lines are drawn among atoms involved in hydrogen bond interactions.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
generally CNS acting drugs, so factors that are important to the
success of CNS drugs were analyzed. In particular, the new mole-
cules should present a good CNS penetration profile and low toxic
effects. Computer predictions were performed with ADMET Pre-
dictor 6.5 [44] and ACD/Percepta 14.0.0 [45] software packages (see
Table S3, Supplementary material).
The lipophilicity (expressed as log P) predicted for 2, 5, 7, 8, 12
and 13 is higher than the traditionally cutoff value of 5 used in drug
design (log P < 5 and/or mlog P < 4.1 [46]). Therefore, these
structures violate the Lipinski’s rules [47]. Moreover, CNS drugs
require more strict rules for a successful penetration to CNS. Thus,
CNS drugs have significantly reduced molecular weights compared
with other therapeutics. Van de Waterbeemd [48] has suggested
that MW should be kept below 450 to facilitate brain penetration
and to be lower than that for oral absorption [49]. All the structures
reported herein show suitable values (MW < 450), with the
exception of the large tetracycles 7e9 and 12e14.
penetration means predicting whether compounds pass across the
bloodebrain barrier. This is crucial in drug design because CNS-
active compounds must pass across it. According to the computed
values [51], 3, 4, 6, 7 and 10 could be CNS inactive due to low brain
penetration, whereas 2, 5, 8, 9 and 11e16 should be good candi-
dates. In particular, the hybrids 2, 5 and 13e16 show a brain
penetration sufficient for CNS activity [52].
Peff quantifies effective permeability across the intestinal
membrane, and characterizes absorption whether by passive
diffusion, active transport, facilitated diffusion, paracellular diffu-
sion, or any other mechanism [53]. MDCK models predict apparent
permeability measured using the MDCK (Madin-Darby Canine
Kidney) [54]. According to the predictions, all the structures show
an adequate permeability to be good candidates (Peff > 0.1,
MDCK > 25), and should be well absorbed compounds (%HIA) [55].
In addition, a middle Caco-2 cell permeability is suggested [56]. The
percent of drug bound with plasma proteins was estimated and the
compounds were predicted to bind weakly to plasma proteins, so it
will be available for diffusion or transport across cell membranes
and thereby finally interact with the target.
As the polarity is involved as the molecule approaches the polar
surface of the cell membrane and desolvates as it moves into the
lipid portion, polar surface area is a key parameter to predict
penetration to CNS. According to Kelder [50] and Waterbeemd [49],
the drugs can be targeted to the CNS with a polar surface area less
The hERG (human Ether-à-go-go Related Gene) encodes potas-
sium channels, which are responsible for the normal repolarization
of the cardiac action potential. Blockage or any other impairment of
these channels in the heart cells can lead to fatal cardiac problems.
Therefore, drug-induced blockage of potassium channels has been
a major concern for the pharmaceutical industry. All the structures,
2
ꢀ
than 60e70 A , a lower range than for other kinds of therapeutics.
Hence, the predicted values for 2e16 fall into the appropriate range.
The bloodebrain barrier (BBB) is a separation of circulating
blood and cerebrospinal fluid in the CNS. Predicting BBB
Fig. 4. Docking pose of inhibitor 15 into hMAO B. (a) The protein structure of hMAO B is rendered as a blue cartoon model. Compound 15 is represented as red sticks. (b) Amino acid
residues of the binding site are color-coded. The flavin adenine dinucleotide cofactor (FAD) and the six water molecules are represented as an integral part of the MAO-B structure
model and are rendered as orange sticks and red balls, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
89
except molecules 3, 4 and 6, show hERG liability [57]. To note that
these three hybrids present the lower lipophilicity of the whole
series. Many drug parameters are affected by lipophilicity. High
lipophilicity frequently leads to compounds with high rapid
metabolic turnover [58] and low solubility and poor absorption. As
lipophilicity (log P) increases, there is an increased probability of
binding to hydrophobic protein targets other than the desired one,
and therefore, there is more potential for toxicity. So, hERG K-
channel blockade is hydrophobic and bind lipophilic substrates
[59]. On other hand, hybrids 3, 4, 7, 8, 12, 13 and 16 could induce
carcinogenicity in chronic mouse studies, whereas none of the
molecules is predicted to present hepatotoxicity [60,61].
75.4, 100.6 or 125.6 MHz, respectively, using solvent peaks [CDCl3:
7.27 (D), 77.2 (C) ppm and DMSO-d6 2.50 (D) and 39.7 (C) ppm] as
internal references. The assignment of chemical shifts was based on
standard NMR experiments (1H, 13C, 1He1H COSY, 1He13C HSQC,
HMBC, DEPT). NMR values (*) can be interchanged. Mass spectra
were recorded on a GC/MS spectrometer with an API-ES ionization
source. Elemental analyses were performed at the IQOG (CSIC,
Spain). TLC analyses were performed on silica F254 and detection
by UV light at 254 nm, or by spraying with phosphomolybdic-
H2SO4 dyeing 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).
To sum up, despite that compounds 13e16 show good brain
penetration profiles, it can be concluded that structure 15 presents
the best drug-like characteristics and ADMET properties of the series.
4.1.2. General procedure for the preparation of hydrochlorides
The hydrochlorides were prepared by dissolving the compounds
in a minimum quantity of ethyl ether, and adding dropwise a
saturated solution of HCl(g) in ethyl ether. A white solid was formed
immediately, that was separated by filtration, washed with ether
and dried.
3. Conclusions
In this paper we have reported the synthesis and pharmaco-
logical evaluation of a series of new donepezileindolyl hybrids as
multifunctional drugs for the potential treatment of AD, using the
multipotent ASS234 hybrid. We have designed amides of type I and
amines of type II (Chart 1), as well as ester 5, carboxylic acid 6 and
alcohol 11 (Chart 2). We have found that, in general, and for the
same substituent, the amines are more potent ChE inhibitors (see
compounds 12,13 versus 7 and 8) or equipotent (see compounds 14,
15 versus 9 and 10) than the corresponding amides, showing a clear
EeAChE inhibition selectivity. For the MAO inhibition activity, am-
ides were not active, and among the amines, compound 14 was
totally MAO A selective, while amines 15 and 16 were quite MAO A
selective. Carboxylic acid derivatives 5 and 11 showed a multi-
potent moderate selective profile as EeACE and MAO A inhibitors.
As a result, we have identified N-((5-(3-(1-benzylpiperidin-4-yl)
propoxy)-1-methyl-1H-indol-2-yl)methyl)prop-2-yn-1-amine
(15), as a potent, in the nanomolar range, inhibitor of MAO A
(IC50 ¼ 5.5 ꢀ 1.4 nM) and moderately potent inhibitor of MAO B
(IC50 ¼ 150 ꢀ 31 nM), AChE (IC50 ¼ 190 ꢀ 10 nM), and BuChE
(IC50 ¼ 830 ꢀ 160 nM).
4.1.3. N-Benzyl-5-methoxy-1-methyl-1H-indole-2-carboxamide (3)
To a solution of known 5-methoxy-1-methyl-1H-indole-2-
carboxylic acid (18), obtained for ester 17 [39] as described [30]
(85 mg, 0.42 mmol) in DMF (3 mL), were added N,N0-dicyclohex-
ylcarbodiimide (DCC) (103.0 mg, 0.5 mmol), hydroxybenzotriazole
(HOBT) (67.6 mg, 0.5 mmol). The reaction mixture was stirred
overnight. Benzylamine (92.0
propylethylamine (DIPEA) (88
m
L, 0.84 mmol) and N,N-di-iso-
m
L, 0.50 mmol) were added, and the
reaction was stirred for 3 h. After complete reaction (tlc analysis),
the solvent was evaporated, and the residue was purified with
column chromatography (1% of MeOH in CH2Cl2) to give amide 3
(73 mg, 60%): mp 201e205 ꢂC; IR (KBr)
n
3287, 2933, 2838, 1636,
1550, 1454, 1423, 1396, 1272, 1239, 1217 cmꢁ1; 1H NMR (500 MHz,
CDCl3)
d
7.38e7.31 (m, 5H, Ph), 7.28 (d, J ¼ 8.7 Hz, H-7), 7.01e6.98
(m, 2H, H-4, H-6), 6.75 (s, H-3), 6.44 (br s, 1H, NH), 4.63 (d,
J ¼ 5.8 Hz, 2H, NHCH2), 4.06 (s, 3H, NCH3), 3.84 (s, OCH3); 13C NMR
(126 MHz, CDCl3)
d
162.3 (CONHCH2Ph), 154.6 (C5), 138.1 (C01, Ph),
Molecular modeling analysis suggests that inhibitor 15 is a
mixed-type EeAChE inhibitor, and that its linear conformation allows
compound 15 to span both the CAS and PAS, contributing to its su-
perior binding toward AChE. We have also observed that the prop-
argylamine group is likely to be an important feature for these
derivatives to exhibit both AChE- and BuChE-inhibitory activities. For
MAO enzymes, docking simulations have shown that the selectivity
of inhibitor 15 for MAO A vs MAO B is likely due to the orientation of
their propargylamine and phenyl moieties in MAO A/B. The pose
obtained in hMAO B for compound 15 showed a preference for the
ligand to be placed in a deeper region, resulting in an energetically
less favorable complex. Compound 15 established more interactions
with the MAO A active site compared to the MAO B active site which
may indicate the 15 interacts more tightly with MAO A.
134.5 (C3a), 131.9 (C2), 128.8, 127.7 (4C, C30,C50,C20,C60, Ph), 127.6
(C40, Ph), 126.1 (C7a), 115.2 (C6), 111.0 (C7), 103.1 (C3), 102.2 (C4),
55.7 (OCH3), 43.5 (NHCH2), 31.7 (NCH3); MS (EI) m/z (%): 146 (54)
[C9H8NO]þ, 161 (100) [C10H11NO]þ, 189 (28) [C11H11NO2]þ, 294 (96)
[M]þ. HRMS (ESI): Calcd for C18H18N2O2: 294.1368. Found:
294.1362. Anal. Calcd. for C18H18N2O2: C, 73.45; H, 6.16; N, 9.52.
Found: C, 73.20; H, 6.04; N, 9.54.
4.1.4. N-Benzyl-1-(5-methoxy-1-methyl-1H-indol-2-yl)
methanamine (4)
To a suspension of LiAlH4 (38.7 mg, 1.02 mmol) in dry THF
(2 mL), at 0 ꢂC, under argon, was added dropwise a solution of the
amide 3 (100 mg, 0.33 mmol) in dry THF (3 mL) and stirring. The
reaction mixture was heated for 5 h. Then, the reaction was cooled
in an ice bath, and water was added carefully. The precipitate was
filtered, the solution was concentrated and the crude, purified by
column chromatography (5% MeOH/CH2Cl2) to afford the amine 4
To sum up, and based on the best observed drug-like charac-
teristics and ADMET properties of the series, compound 15 is a new
and interesting molecule that deserves further analysis as a po-
tential drug for the prevention and treatment of AD.
(75 mg, 79%): mp 101e103 ꢂC; IR (KBr)
n 3436, 3313, 2929, 2829,
1618, 1489, 1452, 1402, 1218 cmꢁ1 1H NMR (500 MHz, CDCl3)
;
4. Experimental part
d
7.37e7.28 (m, 5H, Ph), 7.18 (d, J ¼ 8.8 Hz, 1H, H-7), 7.03 (d,
J ¼ 2.4 Hz, 1H, H-4), 6.85 (dd, J ¼ 8.8, 2.4 Hz, 1H, H-6), 6.33 (s, 1H, H-
3), 3.91 (s, 2H, CH2NH), 3.86 (s, 2H, NCH2Ph), 3.85 (s, 3H, CH3O), 3.73
(s, 3H, CH3eN) [the CH2NHCH2Ph signal was not detected]; 13C
4.1. Chemistry
4.1.1. General methods
NMR (126 MHz, CDCl3) d
154.0 (C5), 140.0 (C10, Ph), 139.0 (C2), 133.2
Melting points were determined in a Koffler apparatus, and are
uncorrected. 1H and 13C NMR spectra were recorded at room
temperature in CDCl3 or DMSO-d6 at 300, 400 or 500 MHz and at
(C7a), 128.4e128.1 (4C, C20,C30,C50,C60, Ph), 127.6 (C04, Ph), 127.0
(C3a), 111.2 (C6), 109.6 (C7), 102.1 (C4), 100.4 (C3), 56.0 (OCH3), 53.1
(NCH2Ph), 45.2 (NHCH2), 29.8 (NCH3); MS (EI) m/z (%): 160 (28)

90
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
[C10H10NO]þ, 175.1 (100) [C11H13NO]þ, 277.1 (44) [C18H17N2O]þ,
280.1 (19) [M]þ. HRMS (ESI): Calcd for C18H20N2O: 280.1576. Found:
280.1575. 4$HCl was prepared using the usual manner to give a
white solid (mp 224e225 ꢂC). Anal. Calcd. for C18H21ClN2O$1/6H2O:
C, 67.60; H, 6.72; N, 8.76. Found: C, 67.62; H, 6.57; N, 9.03.
(32) [Bnepiperidinee(CH2)3]þ, 406 (11) [M]þ; EM (ESI) m/z (%): 407
(M þ 1). HRMS (ESI): Calcd. for C25H30N2O3: 406.2256. Found:
406.2273.
4.1.7. 5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-
2-carbonyl chloride (21)
4.1.5. Ethyl 5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-
indole-2-carboxylate (5)
A solution of acid 6 (91,6 mg, 0.22 mmol) and phosphorous
pentachloride (70.4 mg, 0.34 mmol) in dry dichloromethane
(10 mL) was stirred at reflux for about 2 h. Solvent was removed in
vacuum, the residue treated with a dry ethyl ether and solvent
removed in vacuum again. This operation was repeated twice with
ethyl ether and then twice with chloroform to obtain compound 21
(94.8 mg, 99%) as an oil, that was used immediately, without any
further purification, for the synthesis of the amides 7e10.
To a solution of 1-benzyl-4-(3-chloropropyl)piperidine 19 [22]
(0.419 g, 1.665 mmol) and ethyl 5-hydroxy-1-methyl-1H-indole-
2-carboxylate (20) [31] (0.365 g, 1.665 mmol) in DMF (20 mL),
K2CO3 (0.92 g, 6.656 mmol, 4 equiv) was added. The reaction
mixture was stirred at reflux for 24 h. After complete reaction (tlc
analysis), the solvent was evaporated, the crude 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/
EtOAc,10:1 to 5/1, v/v) to give compound 5 (0.452 g, 63%) as a white
4.1.8. General procedures for the synthesis of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carboxamide
derivatives (7e10)
The crude acyl chloride (21) obtained was dissolved in dry
CH2Cl2 (10 mL) and the solution cooled in an ice-bath. To the so-
lution was added dropwise with stirring, triethylamine (4 equiv)
and an appropriate amine (4 equiv). The mixture was refluxed.
Solvents were removed in vacuum, the residue obtained was pu-
rified by column chromatography. The following compounds were
obtained.
solid: mp 91e94 ꢂC; IR (KBr)
n
3435, 2937, 2917, 1713, 1518, 1471,
7.32e7.29 (m,
1220, 1204, 1087 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
5H, Ph), 7.26 (d, J ¼ 8.9 Hz, 1H, H7-indole), 7.18 (s, 1H, H3-indole),
7.04 (d, J ¼ 1.9 Hz, 1H, H4-indole), 7.01 (dd, J ¼ 8.9, 1.9 Hz, 1H, H6-
indole), 4.36 (q, J ¼ 7.3 Hz, 2H, CO2CH2CH3), 4.04 [s, 3H, N(1)CH3],
3.97 [t, J ¼ 6.6 Hz, 2H, OCH2(CH2)2], 3.49 (s, 2H, NCH2Ph), 2.88 [d,
J ¼ 11.2 Hz, 2H, N(CH2)eq], 1.93 [tm, J ¼ 11.2 Hz, 2H, N(CH2)ax], 1.83e
1.79 (m, 2H, OCH2CH2CH2), 1.70e1.68 [m, 2H, N(CH2)2(CH2)eq],
1.44e1.37 {m, 5H [2H (OCH2CH2CH2) þ 3H (CO2CH2CH3)]}, 1.32e
4.1.9. N-Benzyl-5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-
1H-indole-2-carboxamide (7)
1.25 [m, 3H, N(CH2)2(CH2)axCH]; 13C NMR (125 MHz, CDCl3)
d 162.2
(CO2CH2CH3), 153.9 (C5-indole), 138.6 (C01, Ph), 135.2 (C2-indole),
129.2 (2ꢃ CH, Ph),128.1 (C7a-indole),128.0 (2ꢃ CH, Ph),126.8 (C3a-
indole), 126.0 (C04H, Ph), 119.9 (C6-indole), 111.0 (C7-indole), 109.3
(C3-indole), 103.5 (C4-indole), 68.8 (CH2CH2CH2O), 63.5 (NCH2Ph),
60.4 (CO2CH2CH3), 53.9 [2ꢃ CH2, N(CH2)2(CH2)2CH], 35.6 [CH,
N(CH2)2(CH2)2CH], 32.9 (CH2CH2CH2O), 32.4 [2ꢃ CH2,
N(CH2)2(CH2)2CH], 31.7 [N(1)CH3], 26.7 (CH2CH2CH2O), 14.3
(CO2CH2CH3); MS (EI) m/z (%): 91 (58) [PhCH2]þ, 216 (77) [Bne
Piperidinee(CH2)3]þ, 343 (7) [M ꢁ CH2Ph]þ, 389 (11) [M ꢁ Ph]þ,
434 (67)[M]þ. Anal. Calcd. for C27H34N2O: C, 74.62; H, 7.89; N, 6.45.
Found: C, 74.81; H, 7.94; N, 6.74.
Following the General procedure, reaction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carbonyl
chloride (21) (95.7 mg, 0.22 mmol) with benzylamine (50
mL,
0.45 mmol), and triethylamine (0.12 mL, 0.88 mmol) in dry
dichloromethane (10 mL), after 12 h, and column chromatography
(1% of MeOH in CH2Cl2) gave product 7 (89.3 mg, 80%) as a white
solid: mp 165e167 ꢂC; IR (KBr)
1548, 1465, 1390, 1240, 1205 cmꢁ1
7.37e7.30 (m, 10H, 2ꢃ Ph), 7.27 (d, J ¼ 8.8 Hz, 1H, H7-indole), 6.99
(d, J ¼ 2.4 Hz, 1H, H4-indole), 6.97 (dd, J ¼ 8.8, 2.4 Hz, 1H, H6-
indole), 6.73 (s, H1, H3-indole), 6.40 (t, 5.4 Hz, 1H,
n
3435, 3307, 3082, 2936, 2917, 1637,
;
1H NMR (500 MHz, CDCl3)
d
J
¼
CONHCH2Ph), 4.63 (d, J ¼ 5.4 Hz, 2H, CONHCH2Ph), 4.05 [s, 3H, N(1)
CH3], 3.95 (t, J ¼ 6.6 Hz, 2H, OCH2CH2CH2), 3.49 (s, 2H, NCH2Ph),
2.88 [d, J ¼ 9.7 Hz, 2H, N(CH2)eq], 1.94 [m, 2H, N(CH2)ax], 1.83e1.80
(m, 2H, OCH2CH2CH2), 1.78e1.68 [m, 2H, N(CH2)2(CH2)eq], 1.43e
1.39 (m, 2H, OCH2CH2CH2), 1.28e1.24 [m, 3H, N(CH2)2(CH2)axCH];
4.1.6. 5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-
2-carboxylic acid (6)
To a solution of the ester 5 (1.18 g, 2.7 mmol) in EtOH (10 mL)
was added a solution of lithium hydroxide (129 mg, 5.4 mmol) in
EtOH (3 mL). The reaction mixture was stirred at reflux for 2 h. After
complete reaction, the solvent was evaporated, water was added
and acidified with cc HCl. until pH 6. Then, ethyl acetate was added
and the precipitate filtered, washed with ethyl acetate to afford the
13C NMR (125 MHz, CDCl3)
d 162.3 (CONHCH2Ph),154.0 (C5-indole),
138.1 (2C, 2ꢃ C01, Ph), 134.5 (C7a-indole), 131.9 (C2), 129.2e126.8
(2ꢃ 5CH, 2ꢃ NCH2C6H5),126.2 (C3a-indole),115.7 (C6-indole),110.9
(C7-indole), 103.3 (C4-indole), 103.0 (C3-indole), 68.8
(OCH2CH2CH2), 63.5 (NCH2Ph), 53.9 [2ꢃ CH2, N(CH2)2(CH2)2CH],
43.5 (CONHCH2Ph), 35.6 [N(CH2)2(CH2)2CH], 32.9 (OCH2CH2CH2),
32.3 [2ꢃ CH2, N(CH2)2(CH2)2CH], 31.7 [N(1)CH3], 26.7
(OCH2CH2CH2); MS (EI) m/z (%): 91 (100) [M ꢁ Bn]þ, 172 (63)
corresponding free acid 6 (1.082 g, 98%): mp 104e106 ꢂC; IR (KBr)
3435, 3023, 2936, 1622, 1562, 1516, 1466, 1393, 1286, 1199 cmꢁ1; 1H
NMR (500 MHz, CDCl3) 7.47e7.45 (m, 2H, Ph), 7.38e7.35 (m, 3H,
n
d
Ph), 7.21 (d, J ¼ 8.9 Hz, 1H, H7-indole), 7.18 (s, 1H, 3-indole), 7.02 (d,
J ¼ 2.0 Hz, 1H, H4-indole), 6.90 (dd, J ¼ 8.9, 2.0 Hz, 1H, H6-indole),
4.08 [s, 3H, N(1)CH3], 4.04 (s, 2H, NCH2Ph), 3.93 [t, J ¼ 6.4 Hz, 2H,
OCH2(CH2)2], 3.36 [d, J ¼ 11.3 Hz, 2H, N(CH2)eq], 2.39 [t, J ¼ 11.3 Hz,
[C12H14N]þ, 202 (9) [M
ꢁ
C18H17N2O2],þ 216 (41)
[M C
ꢁ
17H15N2O2]þ, 495 (3) [M]þ. HRMS (ESI): Calcd. for
C
32H37N3O2: 495.2886. Found: 495.2903. Anal. Calcd. for
32H37N3O2: C, 77.54; H, 7.52; N, 8.48. Found: C, 77.17; H, 7.25; N,
2H, N(CH2)ax], 1.81e1.65 (m, 6H, OCH2CH2CH2, N(CH2)2(CH2)eq
,
C
OCH2CH2CH2), 1.45e1.41 [m, 3H, N(CH2)2(CH2)axCH] [the CO2H
8.57.
signal was not detected]; 13C NMR (125 MHz, CDCl3)
d
167.0 (CO2H),
153.3 (C5-indole), 134.8 (2C: C01, Ph; C2-indole), 130.9 (2ꢃ CH, Ph),
128.8 (C7a-indole), 128.7 (2ꢃ CH, Ph), 126.4 (2C: C04, Ph; C3a-
indole), 115.6 (C6-indole), 110.8 (C7-indole), 107.7 (C3-indole),
103.7 (C4-indole), 68.7 (CH2CH2CH2O), 60.8 (NCH2Ph), 51.9 [2ꢃ
CH2, N(CH2)2(CH2)2CH], 34.2 [N(CH2)2(CH2)2CH], 32.2 [CH2e
(CH2)2O], 31.6 [N(1)CH3], 29.7 N(CH2)2(CH2)2CH], 26.5
(CH2CH2CH2O); MS (EI) m/z (%): 91 (100) [PhCH2]þ, 172 (50), 216
4.1.10. 5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-N-phenyl-
1H-indole-2-carboxamide (8)
Following the General procedure, reaction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carbonyl
chloride (21) (95 mg, 0.22 mmol) with aniline (41 mg, 0.44 mmol)
and triethylamine (0.12 mL) in dry dichloromethane (10 mL), after
overnight, and column chromatography (MeOH/CH2Cl2 from 1%

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
91
to 3%) gave product 14 (90.4 g, 84%) as a white solid: mp 189e
192 ꢂC; IR (KBr)
3435, 3294, 3060, 2917, 1644, 1597, 1538, 1499,
1466, 1443, 1316, 1243, 1207 cmꢁ1 1H NMR (500 MHz, CDCl3)
7.84 (s, 1H, NHC6H5), 7.62e7.59 (m, 2H, H02, H06, NHC6H5), 7.39e
chromatography (MeOH/CH2Cl2 from 1% to 3%) gave product 10
(40 mg, 60%) as a white solid: mp 163e165 ꢂC; IR (KBr)
3435, 3291,
3060, 2919, 1638, 1542, 1466, 1391, 1275, 1242, 1206 cmꢁ1; 1H NMR
(500 MHz, CDCl3)
7.32e7.29 (m, 5H, Ph), 7.25 (d, J ¼ 8.8 Hz, 1H,
n
n
;
d
d
7.36 (m, 2H, H03, H05, NHC6H5), 7.32e7.17 (m, 6H, NCH2C6H5, H7-
indole), 7.15 (t, J ¼ 7.3 Hz, 1H, H04, NHC6H5), 7.05 (d, J ¼ 2.4 Hz, 1H,
H4-indole), 7.02 (dd, J ¼ 9.3, 2.4 Hz, 1H, H6-indole), 6.89 (s, 1H,
H3-indole), 4.06 [s, 3H, N(1)CH3], 3.98 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2CH2), 3.50 (s, 2H, NCH2Ph), 2.89 [d, J ¼ 10.8 Hz, 2H,
N(CH2)eq], 1.95 [m, 2H, N(CH2)ax], 1.83e1.80 (m, 2H,
OCH2CH2CH2), 1.71e1.69 [m, 2H, N(CH2)2(CH2)eq], 1.58 (m, 2H,
OCH2CH2CH2), 1.44e1.42 [m, 2H, N(CH2)2(CH2)axCH], 1.30 [m, 1H,
H7-indole), 7.01e6.99 (m, 2H, H4-indole, H6-indole), 6.77 (s, 1H,
H3-indole), 6.29 (s, 1H, CONH), 4.23 (dd, J ¼ 5.4, 2.5 Hz, 2H,
NHCH2C^CH), 4.02 [s, 3H, N(1)CH3], 3.96 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2CH2), 3.50 (s, 2H, NCH2Ph), 2.88 [m, 2H, N(CH2)eq], 2.29 (t,
J ¼ 2.5 Hz, 1H, NHCH2C^CH), 1.96 [m, 2H, N(CH2)ax], 1.82e179 (m,
2H, OCH2CH2CH2),1.70e1.68 [m, 2H, N(CH2)2(CH2)eq],1.43e1.41 (m,
2H, OCH2CH2CH2), 1.29e125 [m, 3H, N(CH2)2(CH2)axCH]; 13C NMR
(126 MHz, CDCl3) d
162.0 (CONH), 154.0 (C5-indole), 138.3 (C01, Ph),
N(CH2)2(CH2)axCH]; 13C NMR (125 MHz, CDCl3)
d
160.4 (CONHPh),
134.6 (C7a-indole), 131.2 (C2-indole), 129.3 (2ꢃ CH, Ph), 128.1 (2ꢃ
CH, Ph), 126.9 (C04, Ph), 126.1 (C3a-indole), 116.0 (C6-indole), 111.0
(C7-indole), 103.6 (C3-indole)*, 103.3 (C4-indole)*, 79.4
(NCH2C^CH), 71.8 (NCH2C^CH), 68.8 (OCH2CH2CH2), 63.4
(NCH2Ph), 53.8 [N(CH2)2(CH2)2CH], 35.5 [N(CH2)2(CH2)2CH], 32.9
(OCH2CH2CH2), 32.2 [N(CH2)2(CH2)2CH], 31.6 [N(1)CH3], 29.2
(NHCH2C^CH), 26.7 (OCH2CH2CH2); MS (EI) m/z (%): 91 (100)
[M ꢁ Bn]þ, 172 (68) [C12H14N]þ, 188 (7), 202 (9) [M ꢁ C14H13N2O2]þ,
216 (35) [M ꢁ C13H11N2O2]þ, 443 (9) [M]þ. HRMS (ESI): Calcd. for
154.1 (C5-indole), 137.7 (2C, 2ꢃ C01, CONHC6H5, NHCH2C6H5),
134.8 (C7a-indole), 132.1 (C2), 129.2 (2ꢃ CH, CONHC6H5), 129.1
(2ꢃ CH, NHCH2C6H5), 128.1 (2ꢃ CH, NHCH2C6H5), 126.9 (C04,
NHCH2C6H5), 126.1 (C3a-indole), 124.4 (C04, CONHC6H5), 119.9
(2ꢃ CH, CONHC6H5), 116.2 (C6-indole), 111.0 (C7-indole), 103.5
(C3-indole), 103.5 (C4-indole), 68.8 (OCH2CH2CH2), 63.4
(NCH2Ph), 53.9 [N(CH2)2(CH2)2CH], 35.5 [N(CH2)2(CH2)2CH], 32.9
(OCH2CH2CH2), 32.3 [N(CH2)2(CH2)2CH], 31.7 [N(1)CH3], 26.7
(OCH2CH2CH2); MS (EI) m/z (%): 91 (100) [M ꢁ Bn]þ, 172 (72)
[C12H14N]þ, 216 (41) [M ꢁ C16H13N2O2]þ. HRMS (ESI): Calcd. for
C
C
28H33N3O2: 443.2573. Found: 443.2577. Anal. Calcd. for
28H33N3O2.1/3H2O: C, 74.80; H, 7.55; N, 9.35. Found: C, 74.89; H,
C
C
31H35N3O2: 481.2729. Found: 481.2729. Anal. Calcd. for
31H35N3O2$1/3H2O: C, 76.35; H, 7.37; N, 8.62. Found: C, 76.68; H,
7.21; N, 9.46.
7.06; N, 8.77.
4.1.13. (5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-
2-yl)methanol (11)
4.1.11. (5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-
2-yl)(morpholino)methanone (9)
Following the General procedure, reaction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carbonyl
Following the General procedure, reaction of ethyl 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carboxylate
5 (221.8 mg, 0.51 mmol) with LiAlH4 (58.1 mg, 1.53 mmol, 3 equiv)
in THF (3 mL), after 15 min, and column chromatography (MeOH/
CH2Cl2 from 1% to 5%) gave product 11 (200 mg, 99%) as a white
chloride (21) (162.0 mg, 0.38 mmol) with morpholine (66
mL,
0.76 mmol) and triethylamine (0.20 mL) in dichloromethane
(10 mL), after 12 h, and column chromatography (1% of MeOH in
CH2Cl2) gave product 9 (130 mg, 72%): as a white solid: mp 125e
solid: mp 93e96 ꢂC; IR (KBr)
n 3435, 3101, 3101, 3065, 2929, 2812,
1620, 1488, 1474, 1196, 1157, 1032 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
7.31e7.29 (m, 5H, Ph), 7.19 (d, J ¼ 8.8 Hz, 1H, H7-indole), 7.03 (d,
128 ꢂC; IR (KBr)
n
3435, 3084, 2921, 2851, 1615, 1532, 1453, 1273,
7.35e7.32 (m, 5H, Ph),
J ¼ 2.4 Hz, 1H, H4-indole), 6.88 (dd, J ¼ 8.8, 2.4 Hz, 1H, H6-indole),
6.35 (s, 1H, H3-indole), 4.77 (s, 2H, CH2OH), 3.96 [t, J ¼ 6.6 Hz, 2H,
OCH2(CH2)2], 3.77 [s, 3H, N(1)CH3], 3.48 (s, 2H, NCH2Ph), 2.87 [d,
J ¼ 11.7 Hz, 2H, N(CH2)eq], 1.93 [tm, J ¼ 11.7 Hz, 2H, N(CH2)ax], 1.81e
1.78 (m, 2H, OCH2CH2CH2), 1.69e1.67 [m, 2H, N(CH2)2(CH2)eq],
1236,1116 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
7.27 (d, J ¼ 9.3 Hz, 1H, H7-indole), 7.05 (d, J ¼ 2.4 Hz,1H, H4-indole),
6.98 (dd, J ¼ 9.3, 2.4 Hz, 1H, H6-indole), 6.52 (s, 1H, H3-indole), 3.99
(t, J ¼ 6.8 Hz, 2H, OCH2CH2CH2), 3.85 [s, 3H, N(1)CH3], 3.84e3.80
[m, 4H, CON(CH2)2(CH2)2O], 3.77e3.71 [m, 4H, CON(CH2)2(CH2)2O],
3.53 (s, 2H, NCH2Ph), 2.92 (dm, J ¼ 10.7 Hz, 2H, N(CH2)eq], 1.98 [m,
2H, N(CH2)ax], 1.86e1.84 (m, 2H, OCH2CH2CH2), 1.83e1.80 [m, 2H,
N(CH2)2(CH2)eq], 1.46e1.44 (m, 2H, OCH2CH2CH2), 1.36e1.32 [m,
1.41e1.39
N(CH2)2(CH2)axCH] (the signal for CH2OH was not detected); 13C
NMR (125 MHz, CDCl3)
153.4 (C5-indole), 139.0 (C01, Ph), 138.6
(m,
2H,
OCH2CH2CH2), 1.28e127
[m,
3H,
d
(C2-indole), 133.5 (C7a-indole), 129.2 (2ꢃ CH, Ph), 128.0 (2ꢃ CH,
Ph), 127.3 (C3a-indole), 126.8 [C0(4)H, Ph], 112.9 (C6-indole), 109.8
(C7-indole), 103.7 (C4-indole), 100.9 (C3-indole), 69.0
(CH2CH2CH2O), 63.5 (NCH2Ph), 57.2 (CH2OH), 53.9 [2ꢃ CH2,
3H, N(CH2)2(CH2)axCH]; 13C NMR (125 MHz, CDCl3)
d 163.2 [CON(-
CH2)2(CH2)2O],153.9 (C5-indole),138.4 (C01, Ph),133.4 (C7a-indole),
131.3 (C2), 129.2 (2ꢃ CH, Ph), 128.1 (2ꢃ CH, Ph), 126.9 (C04, Ph),
126.5 (C3a-indole), 114.9 (C6-indole), 110.6 (C7-indole), 103.5 (C4-
indole), 103.4 (C3-indole), 68.9 (OCH2CH2CH2), 67.0 [2C, CON(-
CH2)2(CH2)2O], 63.5 (NCH2Ph), 53.9 [N(CH2)2(CH2)2CH], 35.5
[N(CH2)2(CH2)2CH], 32.9 (OCH2CH2CH2), 32.3 [N(CH2)2(CH2)2CH],
31.9 [N(1)CH3], 26.7 (OCH2CH2CH2); MS (EI) m/z (%): 172 (100)
N(CH2)2(CH2)2CH],
35.6
[CH,
N(CH2)2(CH2)2CH],
32.9*
(CH2CH2CH2O), 32.3* [2ꢃ CH2, N(CH2)2(CH2)2CH], 29.9 [N(1)CH3],
26.7 (CH2CH2CH2O); MS (EI) m/z (%): 91 (100) [M ꢁ Bn]þ, 172 (68)
[C12H14N]þ, 202 (9) [M
ꢁ
C14H13N2O2]þ, 216 (35)
[M
C
ꢁ
C
13H11N2O2]þ, 392 (9) [M]þ. HRMS (ESI): Calcd. for
[C12H14N]þ, 202 (19) [M
ꢁ
C15H17N2O3]þ, 216 (89)
25H32N2O2: 392.2464. Found: 392.2477. Anal. Calcd. for
25H32N2O2: C, 76.49; H, 8.22; N, 7.14. Found: C, 75.9; H, 7.93; N,
[M
C
C
ꢁ
C
14H15N2O3]þ, 475 (24) [M]þ. HRMS (ESI): Calcd. for
C
29H37N3O3: 475.2835. Found: 475.2842. Anal. Calcd. for
29H37N3O3.1/3H2O: C, 72.32; H, 7.88; N, 8.72. Found: C, 72.37; H,
7.15.
7.56; N, 8.84.
4.1.14. General procedures for the synthesis of (5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)
methanamine derivatives (12e16)
4.1.12. 5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-N-(prop-
2-yn-1-yl)-1H-indole-2-carboxamide (10)
Following the General procedure, reaction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-carbonyl
chloride (21) (63.8 mg, 0.15 mmol) with prop-2-yn-1-amine
A suspension of LiAlH4 in freshly distilled dioxane was cooled at
0
ꢂC and a solution of the amides (7e10) in dioxane was added
dropwise with stirring. The mixture was refluxed for the time
indicated for each case. When the reaction was over, the mixture
was cooled in an ice-bath and, water was added dropwise. The
precipitate was removed by filtration. The solvent was removed,
(20.0
mL g, 0.30 mmol) and triethylamine (83.0 mL, 0.60 mmol) in
dry dichloromethane (10 mL), after 12 h, and column

92
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
and the residue purified by column chromatography to afford the
amines.
[C25H31N2O]þ, 467 (12) [M]þ. HRMS (ESI): Calcd. for C31H37N3O:
467.2937. Found: 467.2935. Bis-chlorhydrate 13$2HCl was prepared
using the usual method to afford a white solid (mp 166e168 ꢂC).
Anal. Calcd. for C31H37N3O$2HCl$1/2H2O: C, 67.75; H, 7.34; N, 7.65.
Found: C, 67.94; H, 7.10; N, 8.07.
4.1.15. N-Benzyl-1-(5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-
methyl-1H-indol-2-yl)methanamine (12)
Following the General procedure, reaction of N-benzyl-5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indole-2-
carboxamide (7) (98 mg, 0.21 mmol) with LiAlH4 (24 mg,
0.63 mmol) in dry dioxane (10 mL), after 7 h, and column chro-
matography (MeOH/CH2Cl2 from 1% to 3%) gave product 12 (60 mg,
4.1.17. 4-((5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-
indol-2-yl)methyl)morpholine (14)
Following the General procedure, reaction of (5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-
63%) as a white solid: mp 74e76 ꢂC; IR (KBr)
2936, 1619, 1491, 1472, 1453, 1393, 1249, 1195, 1160 cmꢁ1; 1H NMR
(500 MHz, CDCl3)
7.36e7.26 (m, 10H, 2Ph), 7.17 (d, J ¼ 8.9 Hz, 1H,
n
3435, 3310, 3025,
yl)(morpholino)methanone (9) (192 mg, 0.40 mmol) with LiAlH4
(46.0 mg, 1.21 mmol) in dry dioxane (8 mL), after 5 h, and column
chromatography (MeOH/CH2Cl2 from 1% to 3%) gave product 14
d
H7-indole), 7.02 (d, J ¼ 2.3 Hz, 1H, H4-indole), 6.83 (dd, J ¼ 8.9,
2.3 Hz, H6-indole), 6.30 (s, H1, H3-indole), 3.97 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2CH2), 3.90 [br s, 2H, C(2)CH2NHCH2Ph]*, 3.85 [s, 2H, C(2)
CH2NHCH2Ph]*, 3.72 [s, 3H, N(1)CH3], 3.52 (s, 2H, NCH2Ph), 2.90 [d,
J ¼ 10.1 Hz, 2H, N(CH2)eq], 1.97 [m, 2H, N(CH2)ax], 1.83e1.76 (m, 2H,
OCH2CH2CH2), 1.71e1.69 [m, 2H, N(CH2)2(CH2)eq], 1.43e1.41 (m, 2H,
OCH2CH2CH2), 1.39e1.30 [m, 3H, N(CH2)2(CH2)axCH] (the signal for
C(2)CH2NHCH2Ph could not be detected); 13C NMR (125 MHz,
(121 mg, 65%) as a white solid: mp 113e115 ꢂC; IR (KBr)
n
3436,
3029, 2938, 2905, 2850, 2804, 2761, 1621, 1489, 1467, 1452, 1201,
1114 cmꢁ1; 1H NMR (500 MHz, CDCl3)
7.33e7.29 (m, 5H, Ph), 7.17
d
(d, J ¼ 8.8 Hz,1H, H7-indole), 7.01 (d, J ¼ 2.5 Hz,1H, H4-indole), 6.84
(dd, 1H, H6-indole), 6.27 (s, 1H, H3-indole), 3.96 (t, J ¼ 6.8 Hz, 2H,
OCH2CH2CH2), 3.76 [s, 3H, N(1)CH3], 3.67 [m, 4H, N(CH2)2(CH2)2O],
3.59 [s, 2H, C(2)CH2N(CH2)2(CH2)2O)], 3.50 (s, 2H, NCH2Ph), 2.88 (d,
J ¼ 10.3 Hz, 2H, N(CH2)2(CH2)eq], 2.45 (m, 4H, N(CH2)2(CH2)2O], 1.94
[m, 2H, N(CH2)ax], 1.81e1.78 (m, 2H, OCH2CH2CH2), 1.76e1.68
[N(CH2)2(CH2)eq], 1.43e1.39 (m, 2H, OCH2CH2CH2), 1.29 [m, 3H,
CDCl3)
d
153.3 (C5-indole), 140.1 (2C, 2C01, Ph), 138.9 (C7a-indole),
133.2 (C2), 129.3, 128.4, 128.1, 127.6 (10C, NCH2C6H5, NHCH2C6H5),
127.0 (C3a-indole), 111.8 (C6-indole), 109.5 (C7-indole), 103.3 (C4-
indole), 100.4 (C3-indole), 69.0 (OCH2CH2CH2), 63.7 (NCH2Ph),
53.8 [C(2)CH2NHCH2Ph]*, 53.2 [2CH2, N(CH2)2(CH2)2CH]*, 45.2
[C(2)CH2NHCH2Ph]*, 35.5 [N(CH2)2(CH2)2CH], 32.8 (OCH2CH2CH2),
32.1 [2ꢃ CH2, N(CH2)2(CH2)2CH], 30.0 [N(1)CH3], 26.7
(OCH2CH2CH2); MS (EI) m/z (%): 172 (46), 216 (37), 283 (14), 374
(100), 383 (13), 481 (11) [M]þ. HRMS (ESI): Calcd. for C32H39N3O:
481.3093. Found: 481.3111. Bis-chlorhydrate 12$2HCl was prepared
using the usual method to afford a white solid (mp 245e248 ꢂC).
Anal. Calcd. for C32H39N3O$2HCl$3/2H2O: C, 66.08; H, 7.63; N, 7.22.
Found: C, 66.06; H, 7.07; N, 7.42.
N(CH2)2(CH2)axCH]; 13C NMR (125 MHz, CDCl3)
d 153.3 (C5-indole),
136.3 (2C, C01, Ph; C2-indole), 133.3 (C7a-indole), 129.2 (2ꢃ CH, Ph),
128.1 (2ꢃ CH, Ph), 127.4 (2C, C04, Ph; C3a-indole), 112.0 (C6-indole),
109.5 (C7-indole), 103.3 (C4-indole), 102.1 (C3-indole), 69.1
(OCH2CH2CH2), 67.0 [2C, N(CH2)2(CH2)2O], 63.5 (NCH2Ph), 55.3
[C(2)CH2N(CH2)2(CH2)2O)], 53.9 [2CH2, N(CH2)2(CH2)2CH], 53.4 [2C,
N(CH2)2(CH2)2O], 35.5 [N(CH2)2(CH2)2CH], 32.9 (OCH2CH2CH2),
32.3 [2C, N(CH2)2(CH2)2CH], 29.9 [N(1)CH3], 26.8 (OCH2CH2CH2);
MS (EI) m/z (%): 172 (86) [C12H14N]þ, 188 (12) [C13H18N]þ, 202 (10)
[C14H20N]þ, 216 (89) [C15H22N]þ, 375 (100) [C25H31N2O]þ, 461 (51)
[M]þ. HRMS (ESI): Calcd. for C29H39N3O2: 461.3042. Found:
461.3047. Bis-chlorhydrate 14$2HCl was prepared using the usual
method to afford a white solid (mp 249e252 ꢂC). Anal. Calcd. for
4.1.16. N-((5-(3-(1-Benzylpiperidin-4-yl)propoxy)-1-methyl-1H-
indol-2-yl)methyl)aniline (13)
C29H39N3O2$2HCl$3/2H2O: C, 62.02; H, 7.90; N, 7.48. Found: C,
Following the General procedure, reaction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-N-phenyl-1H-indole-2-
carboxamide (8) (174 mg, 0.36 mmol) with LiAlH4 (41.2 mg,
1.1 mmol) in dry dioxane (7 mL), after 5 h, and column chroma-
tography (MeOH/CH2Cl2 from 1% to 3%) gave product 13 (123 mg,
62.07; H, 7.20; N, 7.75.
4.1.18. Reduction of 5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-
methyl-N-(prop-2-yn-1-yl)-1H-indole-2-carboxamide (10)
Following the General procedure, reduction of 5-(3-(1-
benzylpiperidin-4-yl)propoxy)-1-methyl-N-(prop-2-yn-1-yl)-1H-
indole-2-carboxamide (10) (50 mg, 0.11 mmol) with LiAlH4
(12.8 mg, 0.33 mmol) in dry THF (5 mL), after 8 h, and column
chromatography (MeOH/CH2Cl2 from 1% to 3%) gave products 15
(24.7 mg, 51%) and 16 (14.1 mg, 29%). N-((5-(3-(1-Benzylpiperidin-4-
yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)prop-2-yn-1-amine
73%) as a white solid: mp 114e116 ꢂC; IR (KBr)
2905, 1624, 1599, 1501, 1476, 1424, 1312, 1250, 1197, 1026 cmꢁ1; 1H
NMR (500 MHz, CDCl3) 7.32e7.27 (m, 5H, NCH2C6H5), 7.25e7.22
n 3412, 3022, 2941,
d
(m, 2H, H02, H06, CH2NHC6H5), 7.19 (d, J ¼ 8.8 Hz, 1H, H7-indole),
7.03 (d, J ¼ 2.4 Hz, 1H, H4-indole), 6.87 (dd, J ¼ 8.8, 2.4 Hz, 1H, H6-
indole), 6.78 (m, 1H, H04, CH2NHC6H5), 6.71 (m, 2H, H03, H05,
CH2NHC6H5), 6.39 (s, 1H, H3-indole), 4.39 [d, J ¼ 4.9 Hz, 2H, C(2)
CH2NHPh], 3.97 (t, J ¼ 6.6 Hz, 2H, OCH2CH2CH2), 3.72 [s, 3H, N(1)
CH3], 3.50 (s, 2H, NCH2Ph), 2.89 (d, J ¼ 11.2 Hz, 2H, N(CH2)eq], 2.05e
1.93 [m, 2H, N(CH2)ax], 1.82e1.80 [m, 2H, OCH2CH2CH2), 1.79e1.69
[m, 2H, N(CH2)2(CH2)eq], 1.44e1.40 (m, 2H, OCH2CH2CH2), 1.30e
1.26 [m, 3H, N(CH2)2(CH2)axCH] (the signal for C(2)CH2NHPh could
(15): mp 89e92 ꢂC; IR (KBr)
1490, 1472, 1451, 1198 cmꢁ1; 1H NMR (500 MHz, CDCl3)
n
3436, 3301, 3269, 2919, 2862, 1621,
7.33e7.26
d
(m, 5H, Ph), 7.17 (d, J ¼ 8.8 Hz, 1H, H7-indole), 7.02 (d, J ¼ 2.3 Hz, 1H,
H4-indole), 6.84 (dd, J ¼ 8.8, 2.3 Hz, 1H, H6-indole), 6.33 (s, 1H, H3-
indole), 4.00 [br s, 2H, C(2)CH2NHCH2C^CH], 3.96 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2CH2), 3.73 [s, 3H, N(1)CH3], 3.54 (s, 2H, NCH2Ph), 3.45 [d,
J ¼ 2.4 Hz, 2H, C(2)CH2NHCH2C^CH], 2.92 [d, J ¼ 10.4 Hz, 2H,
N(CH2)eq], 2.27 [t, J ¼ 2.4 Hz,1H, C(2)CH2NHCH2C^CH],1.99 (m, 2H,
N(CH2)ax], 1.79e1.77 [m, 2H, OCH2CH2CH2), 1.76e1.72 [m, 2H,
N(CH2)2(CH2)eq],1.43e1.41 (m, 2H, OCH2CH2CH2),1.39e1.32 [m, 3H,
N(CH2)2(CH2)axCH)] (the signal for C(2)CH2NHCH2C^CH could not
not be detected); 13C NMR (125 MHz, CDCl3)
d 153.5 (C5-indole),
147.8 (C001, CH2NHC6H5), 138.7 (C01, NCH2C6H5), 137.5 (C2), 133.2
(C7a-indole), 129.3 (2ꢃ CH, C003, C005, CH2NHC6H5), 129.2 (2ꢃ CH,
NCH2C6H5), 128.1 (2ꢃ CH, NCH2C6H5), 127.6 (C04, NCH2C6H5), 126.8
(C3a-indole), 118.3 [CH, C004, CH2NHC6H5], 113.0 (2ꢃ CH, C002, C006,
CH2NHC6H5), 112.4 (C6-indole), 109.6 (C7-indole), 103.5 (C4-
indole), 100.8 (C3-indole), 69.1 (OCH2CH2CH2), 63.5 (NCH2Ph),
53.9 [2CH2, N(CH2)2(CH2)2CH], 41.0 [C(2)CH2NHPh], 35.5
[N(CH2)2(CH2)2CH], 32.9 (OCH2CH2CH2), 32.1 [2ꢃ CH2,
N(CH2)2(CH2)2CH], 30.1 [N(1)CH3)], 26.8 (OCH2CH2CH2); MS (EI) m/
z (%): 160 (15), 172 (23) [C12H14N]þ, 216 (17) [C15H22N]þ, 375 (100)
be detected); 13C NMR (126 MHz, CDCl3)
d 153.5 (C5-indole), 137.8
(2C, C01, C6H5; C2-indole), 133.2 (C7a-indole), 129.4 (2xCH, Ph),
128.2 (2xCH, Ph), 127.5 (2C, C04, Ph; C3a-indole), 112.0 (C6-indole),
109.5 (C7-indole), 103.3 (C4-indole), 100.9 (C3-indole), 81.8
(NCH2C^CH), 76.6 (NCH2C^CH), 69.0 (OCH2CH2CH2), 63.7
(NCH2Ph),
53.7
[2CH2,
N(CH2)2(CH2)2CH],
44.1
[C(2)

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
93
CH2NCH2C^CH], 37.1 (NCH2C^CH), 35.3 [N(CH2)2(CH2)2CH], 32.8
(OCH2CH2CH2), 32.0 [N(CH2)2(CH2)2CH], 29.7 [N(1)CH3], 26.7
(OCH2CH2CH2); MS (EI) m/z (%): 172 (100) [C12H14N]þ, 188 (12)
[C13H18N]þ, 202 (13) [C14H20N]þ, 216 (83) [C15H22N]þ, 374 (79)
[C25H30N2O]þ, 429 (33) [M]þ. HRMS (ESI): Calcd. for C28H35N3O:
429.278. Found: 429.2801. Bis-chlorhydrate 15$2HCl was prepared
using the usual method to afford a white solid (mp 204e205 ꢂC).
Anal. Calcd. for C28H35N3O$2HCl$4/5H2O: C, 64.05; H, 7.58; N, 8.09.
Found: C, 64.15; H, 7.09; N, 8.25. N-((5-(3-(1-Benzylpiperidin-4-yl)
propoxy)-1-methyl-1H-indol-2-yl)methyl)prop-2-en-1-amine (16):
(version 3.0). Data are expressed as mean ꢀ SEM of at least three
different experiments performed in triplicate.
4.2.2. Monoamine oxidase activity
Monoamine oxidase activities from recombinant human MAO A
and MAO B (SigmaeAldrich, Madrid, Spain) were performed using
a fluorometric method [33]. Tyramine hydrochloride was used as
substrate for both enzymes in a 96-well black opaque microplates
(OptiPlate-96F, PerkinElmer) in a final volume of 200
mL. Serial
dilutions of each inhibitor were pre-incubated for 30 min at 37 ꢂC
with 0.36 U/mL hMAO A or 0.0675 U/mL hMAO B. Following the
mp 57e60 ꢂC; IR (KBr)
n
3435, 3076, 3060, 2912, 1620, 1490, 1473,
7.32e7.29 (m, 5H,
1452, 1203 cmꢁ1 1H NMR (500 MHz, CDCl3)
;
d
pre-incubations, enzymatic reactions were started by adding 100
of a mixture containing 1 mM tyramine, 0.04 U/ml horseradish
peroxidase (HRP) and 25 M Amplex UltraRed reagent in 0.25 mM
ml
Ph), 7.16 (d, J ¼ 8.8 Hz, 1H, H7-indole), 7.01 (d, J ¼ 2.5 Hz, 1H, H4-
indole), 6.83 (dd, J ¼ 8.8, 2.5 Hz, 1H, H6-indole), 6.28 (s 1H, H3-
indole), 5.91 (m, 1H, NCH2CH]CH2), 5.21 dd, J ¼ 17.1, 1.5 Hz, 1H,
NCH2CH]CH2), 5.12 (dd, J ¼ 10.3,1.5 Hz,1H, NCH2CH]CH2), 3.96 (t,
J ¼ 6.6 Hz, 2H, OCH2CH2CH2), 3.89 [s, 2H, C(2)CH2NHCH2CH]CH2],
3.73 [s, 3H, N(1)CH3], 3.51 (br s, 2H, NCH2Ph), 3.32 (m, 2H,
NHCH2CH]CH2), 2.90 [d, J ¼ 10.2 Hz, 2H, N(CH2)eq], 1.97 [m, 2H,
N(CH2)ax], 1.81e1.78 (m, 2H, OCH2CH2CH2), 1.70e1.68 [m, 2H,
N(CH2)2(CH2)eq], 1.59 (br s, 1H, NHCH2CH]CH2), 1.42e1.40 (m, 2H,
OCH2CH2CH2), 1.30 [m, 3H, N(CH2)2(CH2)axCH]; 13C NMR (126 MHz,
m
sodium phosphate (pH 7.4) as final concentrations. The fluores-
cence production associated with peroxidase-coupled production
of resorufin from Amplex UltraRed was continuously measured for
at least one hour at 530 nm in a spectrophotometric plate reader
(FluoStar OPTIMA, BM G Labtech). Control experiments were car-
ried out simultaneously by replacing the inhibitors by distilled
water. In addition, the possible capacity of compounds to modify
the fluorescence generated in the reaction mixture due to non-
enzymatic inhibition was determined by adding these com-
pounds to solutions containing only the Amplex UltraRed reagent
in a sodium phosphate buffer. Substrate-free samples were used as
blanks. Doseeresponse curves were plotted by using the GraphPad
‘PRISM’ software (version 3.0) and IC50 values were accordingly
calculated. Data are expressed as mean ꢀ SEM of at least three
different experiments performed in triplicate.
CDCl3)
d 153.3 (C5-indole), 139.0 (C2-indole), 136.6 (NHCH]CH2),
133.2 (C7a-indole), 129.3 (C01, Ph), 128.1 (2ꢃ CH, Ph), 127.6 (C3a-
indole), 126.9 (2ꢃ CH, Ph), 116.2 (NHCH]CH2), 111.9 (C6-indole),
109.5 (C7-indole), 103.4 (C4-indole), 100.2 (C3-indole), 69.1
(OCH2CH2CH2), 63.4 (NCH2Ph), 53.8 [2CH2, N(CH2)2(CH2)2CH], 51.7
[C(2)CH2NCH2C]CH2],
45.1
[C(2)CH2NCH2CH]CH2],
35.5
[N(CH2)2(CH2)2CH], 32.9 (OCH2CH2CH2), 32.2 [N(CH2)2(CH2)2CH],
29.8 [N(1)CH3], 26.8 (OCH2CH2CH2); MS (EI) m/z (%): 172 (52)
[C12H14N]þ, 188 (7) [C13H18N]þ, 202.2 (6) [C14H20N]þ, 216 (45)
[C15H22N]þ, 374 (100) [C25H30N2O]þ, 431 (24) [M]þ; HRMS (ESI):
Calcd. for C28H37N3O: 429.2937. Found: 429.2934. Bis-chlorhydrate
16$2HCl was prepared using the usual method to afford a white
solid (mp 225e226 ꢂC). Anal. Calcd. for C28H37N3O$2HCl$3/5H2O: C,
62.91; H, 7.98; N, 7.86. Found: C, 62.72; H, 7.24; N, 8.19.
4.3. Molecular modeling
4.3.1. Molecular docking into AChE and BuChE
Compound 15 was assembled as hydrochloride and free base
within Discovery Studio, version 2.1, software package, using
standard bond lengths and bond angles. With the CHARMm force
field [62] and partial atomic charges, the molecular geometry of 15
was energy-minimized using the adopted-based NewtoneRaphson
algorithm. Structures were considered fully optimized when the
energy changes between iterations were less than 0.01 kcal/mol
[63].
4.2. Pharmacology
4.2.1. Cholinesterase activity
Cholinesterase activities were assessed following a spectro-
photometric method [32] using AChE from Electrophorus elec-
tricus (type V-S) and BuChE from equine serum (lyophilized
powder) (SigmaeAldrich, Madrid, Spain). Donepezil and ASS234
were used as standard compounds. Enzymatic reactions took
4.3.2. Molecular docking of compound 15 into EeAChE
The coordinates of E. electricus AChE (PDB ID: 1C2B), were ob-
tained from the Protein Data Bank (PDB). For docking studies, initial
protein was prepared by removing all water molecules, hetero-
atoms, any co-crystallized solvent and the ligand. Proper bonds,
bond orders, hybridization and charges were assigned using pro-
tein model tool in Discovery Studio, version 2.1, software package.
CHARMm force field was applied using the receptoreligand in-
teractions tool in Discovery Studio, version 2.1, software package.
Docking calculations were performed with the program Autodock
Vina [34]. AutoDockTools (ADT; version 1.5.4) was used to add
hydrogens and partial charges for proteins and ligands using Gas-
teiger charges. Flexible torsions in the ligands were assigned with
the AutoTors module, and the acyclic dihedral angles were allowed
to rotate freely. Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75, Trp86,
and Tyr341 receptor residues were selected to keep flexible during
docking simulation using the AutoTors module. Because VINA uses
rectangular boxes for the binding site, the box center was defined
and the docking box was displayed using ADT. For E. electricus AChE
(PDB ID: 1C2B) the docking procedure was applied to whole protein
target, without imposing the binding site (“blind docking”). Using
the GridBox option, the 3-dimensional parameters for docking the
ligand to the protein were determined; the grid center coordinates
place in 96-well plates in a final volume of 300
mL containing
0.1 M phosphate buffer (pH 8.0), 0.035 U/ml AChE or 0.05 U/mL
BuChE and 0.35 mM of 5,50-dithiobis-2-nitrobenzoic acid (DTNB,
SigmaeAldrich, Madrid, Spain). Inhibition curves were plotted by
pre-incubating this mixture with serial dilutions of each com-
pound for 20 min at 37 ꢂC. The activity in absence of compound
was always performed to determine the 100% of enzyme activity.
At the end of the pre-incubation period, 50 mL of substrate were
added to a final concentration of 0.35 mM acetylthiocholine io-
dide or 0.5 mM butyrylthiocholine iodide (SigmaeAldrich).
Enzymatic reactions were allowed for 5 min incubation for AChE
and 25 min for BuChE as the yellowish anion 5-thio-2-
nitrobenzoic acid is continuously produced due to the DTNB
oxidation along with the enzymatic degradation of both sub-
strates. Changes in absorbance were detected at 405 nm in a
spectrophotometric plate reader (FluoStar OPTIMA, BMG Lab-
tech). Color generation is reduced as the enzymatic activities are
inhibited by the compounds and IC50 values were calculated at
the compound concentration that produces 50% of enzymatic
activity inhibition by using the GraphPad ‘PRISM’ software

94
O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
were x ¼ 21.5911, y ¼ 87.752, z ¼ 23.591 and the size coordinates
Appendix A. Supplementary material
ꢀ
were x ¼ 60, y ¼ 60, z ¼ 72 with grid points separated 1 A. Default
parameters were used except num_modes, which was set to 40.
The AutoDock Vina docking procedure used was previously vali-
dated [64].
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.028.
References
4.3.3. Molecular docking of inhibitor 15 into eqBuChE
[1] M. Goedert, M.G.A. Spillantini, Science 314 (2006) 777e781.
[2] I. Grundke-Iqbal, K. Iqbal, Y.C. Tung, M. Quinlan, H.M. Wisniewski, L.I. Binder,
Proc. Natl. Acad. Sci. U. S. A. 93 (1986) 4913e4917.
[3] A. Gella, N. Durany, Cell. Adhes. Migr. 3 (2009) 88e93.
[4] V.N. Talesa, Mech. Ageing Dev. 122 (2001) 1961e1969.
[5] J. Birks, E.J. Grimley, V. Iakovidou, Cochrane Database Syst. Rev. (2000)
CD001191.
The eqBuChE model has been retrieved from the SWISS-
MODEL Repository. This is
a database of annotated three-
dimensional comparative protein structure models generated by
the fully automated homology-modeling pipeline SWISS-MODEL.
A putative three-dimensional structure of eqBuChE has been
created based on the crystal structure of hBuChE (PDB ID: 2PM8),
these two enzyme exhibited 89% sequence identity. Proper bonds,
bond orders, hybridization and charges were assigned using pro-
tein model tool in Discovery Studio, version 2.1, software package.
CHARMm force field was applied using the receptoreligand in-
teractions tool in Discovery Studio, version 2.1, software package.
Docking calculations were performed following the same protocol
described before for EeAChE. All dockings were performed as
[6] M. Racchi, M. Mazzucchelli, E. Porrello, C. Lanni, S. Govoni, Pharm. Res. 50
(2004) 441e451.
[7] R. León, A.G. García, J. Marco-Contelles, Med. Res. Rev. 33 (2013) 139e189.
[8] K.L. Davis, P. Powchick, Lancet 345 (1995) 625e630.
[9] C.M. Spencer, S. Noble, Drugs Aging 13 (1998) 391e400.
[10] E.L. Barner, S.L. Gray, Ann. Pharmacother. 32 (1998) 70e77.
[11] M.B. Youdim, D. Edmonson, K.F. Tipton, Nat. Rev. Neurosci. 7 (2006) 295e309.
[12] P. Riederer, W. Danielczyk, E. Grünblatt, Neurotoxicology 25 (2004) 271e277.
[13] N. Pizzinat, N. Copin, C. Vindis, A. Parini, C. Cambon, Naunyn Schmiedebergs
Arch. Pharmacol. 359 (1999) 428e431.
ꢀ
[14] B.S. Kristal, A.D. Conway, A.M. Brown, J.C. Jain, P.A. Ulluci, S.W. Li, J.W. Burke,
Free Radical Biol. Med. 30 (2001) 924e931.
blinds dockings where a cube of 75 A with grid points separated
ꢀ
1 A, was positioned at the middle of the protein (x ¼ 29.885;
[15] J. Sterling, Y. Herzig, T. Goren, N. Finkelstein, D. Lerner, W. Goldenberg,
I. Miskolczi, S. Molnar, F. Rantal, T. Tamas, G. Toth, A. Zagyva, A. Zekany,
G. Lavian, A. Gross, R. Friedman, M. Razin, W. Huang, B. Krais, M. Chorev,
M.B. Youdim, M. Weinstock, J. Med. Chem. 45 (2002) 5260e5279.
[16] M. Yogev-Falach, O. Bar-Am, T. Amit, O. Weinreb, M.B. Youdim, FASEB J. 20
(2006) 2177e2179.
[17] M.B. Youdim, M. Weinstock, Cell. Mol. Neurobiol. 21 (2002) 555e573.
[18] O. Bar-Am, T. Amit, O. Weinreb, M.B. Youdim, S. Mandel, J. Alzheimers Dis. 21
(2010) 361e371.
y ¼ ꢁ54.992; z ¼ 58.141). Default parameters were used except
num_modes, which was set to 40. The lowest docking-energy
conformation was considered as the most stable orientation.
Finally, the docking results generated were directly loaded into
Discovery Studio, version 2.1.
4.3.4. Molecular docking of compound 15 into human MAO A/B
Compound 15 was assembled as non-protonated amine within
Discovery Studio, version 2.1, software package, following the
procedure described before for cholinesterases. The crystal struc-
tures of human MAO A in complex with harmine (PDB ID 2Z5X) and
human MAO B in complex with safinamide (PDB ID 2V5Z) were
obtained from the Protein Data Bank. For docking studies initial
proteins were prepared. First, in the PDB crystallographic structures
any co-crystallized solvent and the ligand were removed. Then,
proper bonds, bond orders, hybridization and charges were
assigned using protein model tool in Discovery Studio, version 2.1,
software package. CHARMm force field was applied using the re-
ceptoreligand interactions tool in Discovery Studio, version 2.1,
software package. Six water molecules located around the FAD
cofactor were considered in the docking experiments because of
their well-known role into the MAO’s inhibition. Finally, atoms of
the FAD cofactor were defined in their oxidized state. Docking
calculations were performed following the same protocol described
before for EeAChE. For MAO-A, the grid center coordinates were
x ¼ 54.181, y ¼ 160.270, z ¼ 22.052 and the size coordinates were
[19] F.O. Brühlmann, P.-A. Carrupt, B. Testa, M. Catto, F. Leonetti, C. Altomare,
A. Carotti, J. Med. Chem. 44 (2001) 3195e3198.
[20] A. Cavalli, M.L. Bolognesi, M. Minarini, V. Rosini, M. Tumiatti, C. Recanatini,
C. Melchiorre, J. Med. Chem. 51 (2008) 347e372.
[21] A. Samadi, M. Chioua, I. Bolea, C. de los Ríos, I. Iriepa, I. Moraleda, A. Bastida,
G. Esteban, M. Unzeta, E. Gálvez, J. Marco-Contelles, Eur. J. Med. Chem. 46
(2011) 4665e4668.
[22] I. Bolea, J. Juárez-Jiménez, C. de los Ríos, M. Chioua, R. Pouplana, F.J. Luque,
M. Unzeta, J. Marco-Contelles, A. Samadi, J. Med. Chem. 54 (2011) 8251e8270.
[23] V. Pérez, J.L. Marco, E. Fernández-Álvarez, M. Unzeta, Br. J. Pharmacol. 127
(1999) 869e876.
[24] B. Cutillas, S. Ambrosio, M. Unzeta, Neurosci. Lett. 329 (2002) 165e168.
[25] G. Prat, V. Pérez, A. Rubí, M. Casas, M. Unzeta, J. Neural. Transm. 107 (2000)
409e417.
[26] I. Bolea, A. Gella, L. Monjas, C. Pérez, M.I. Rodríguez-Franco, J. Marco-Contelles,
A. Samadi, M. Unzeta, Curr. Alzheimer Res. 10 (2013) 797e808.
[27] G. La Regina, R. Silvestri, V. Gatti, A. Lavecchia, E. Novellino, O. Befani, P. Turini,
E. Agostinelli, Bioorg. Med. Chem. 16 (2008) 9729e9740.
[28] M.L. Wu, J.F. Deng, J. Chin. Med. Assoc. 72 (2009) 446e449.
[29] C. Fernández-García, J.L. Marco, E. Fernández-Álvarez, Tetrahedron 48 (1992)
6917e6928.
[30] M.A. Cruces, C. Elorriaga, E. Fernández-Álvarez, O. Nieto López, O. Eur, J. Med.
Chem. 25 (1990) 257e265.
[31] P.C. Unangst, D.Y. Connor, S.R. Miller, R. Steven, J. Heterocycl. Chem. 33 (1996)
1627e1630.
[32] G.L. Ellman, K.D. Courtney, B.J. Andrés, R.M. Featherstone, Biochem. Pharma-
col. 7 (1961) 88e95.
[33] M. Zhou, N. Panchuk-Valashina, Anal. Biochem. 253 (1997), 169e174.
[34] O. Trott, A.J. Olson, J. Comput. Chem. 31 (2010) 455e461.
[35] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, Bioinformatics 22 (2006) 195e201.
[36] F. Kiefer, K. Arnold, M. Künzli, L. Bordoli, T. Schwede, Nucleic Acids Res. 37
(2009) D387eD392.
[37] M.C. Peitsch, Biotechnology 13 (1995) 658e660.
[38] D.E. Edmonson, A. Mattevi, C. Binda, M. Li, F. Hubálek, Curr. Med. Chem. 11
(2004) 1983e1993.
ꢀ
x ¼ 50, y ¼ 40, z ¼ 40 with grid points separated 1 A; for MAO-B, the
grid center coordinates were x ¼ 55,743, y ¼ 161.175, z ¼ 19.420 and
the size coordinates were x ¼ 46, y ¼ 40, z ¼ 40 with grid points
ꢀ
separated 1 A. Default parameters were used except num modes,
which was set to 40. According to Vina best scored poses, the most
stable complex configurations were considered. The docked ligand
output files was viewed and atomic distances and interactions were
analyzed using Discovery Studio.
[39] T.Z.E. Jones, D. Balsa, M. Unzueta, R.R. Ramsay, J. Neural. Transm. 114 (2007)
707e712.
[40] J. Wang, D.E. Edmonson, Biochemistry 50 (2011) 7710e7717.
[41] R.A. Scerrer, A.J. Leo, Mol. Inform. 30 (2011) 386.
Acknowledgments
[42] A. Samadi, C. de los Ríos, I. Bolea, M. Chioua, I. Iriepa, I. Moraleda, M. Bartolini,
B. Andrisano, E. Gálvez, C. Valderas, M. Unzeta, J. Marco-Contelles, Eur. J. Med.
Chem. 52 (2012) 251e262.
[43] J. Ma, M. Yoshimura, E. Yamashita, A. Nakagawa, A. Ito, T. Tsukihara, J. Mol.
Biol. 338 (1) (2004) 103e114.
[44] AMET Predictor, v.6.5, Simulations Plus, Inc., Lancaster, CA, 2013.
[45] ACD/Percepta 14.0.0, Advanced Chemistry Development, 2013.
[46] I. Moriguchi, S. Hirono, Q. Liu, I. Nakagome, Y. Matsushita, Chem. Pharm. Bull.
40 (1992) 127e130.
This work was supported by the COST action CM1103. KN
acknowledge project supported by the Ministry of Education and
Science of the Republic of Serbia, Contract #172033. JMC and MU
thank MINECO (Spain) for support (SAF2008-07271; SAF2012-
33304). OMBA thanks MINECO (Spain) for a fellowship (BES-2010-
037196).

O.M. Bautista-Aguilera et al. / European Journal of Medicinal Chemistry 75 (2014) 82e95
95
[47] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Delivery Rev.
46 (2001) 3e26.
[57] L.S. Gold, N.B. Manley, T.H. Slone, L. Rohrbach, Environ. Health Perspect. 107
(Suppl. 4) (1999) 527e600.
[48] H. Van de Waterbeemed, F. Atkinson, S. Cole, C. Green, Curr. Med. Chem Cent.
Nerv. Syst. Agents 2 (2002) 229e240.
[58] H. Van de Waterbeemd, D.A. Smith, K. Beaumont, D.K. Walker, J. Med. Chem.
44 (2001) 1313e1333.
[49] H. Van de Waterbeemd, G. Camenish, G. Folkers, J.R. Chretien, O.A. Raevsky,
J. Drug Target. 6 (1998) 151e165.
[50] J. Kelder, P.D.J. Grootenhuis, D.M. Bayada, L.P.C. Delbressine, J.-P. Ploemen,
Pharm. Res. 16 (1999) 1514e1519.
[51] P.A. Nielsen, O. Andersson, S.H. Hansen, K.B. Simonsen, G. Andersson, Drug.
Discov. Today 16 (2011) 472e475.
[52] X. Ma, C. Chen, J. Yang, Acta Pharmacol. Sin. 26 (2005) 500e512.
[53] A. Sjöberg, M. Lutz, C. Tannergren, C. Wingolf, A. Borde, A.L. Ungell, Eur. J.
Pharm. Sci. 48 (2013) 166e180.
[59] S. Ekins, Drug. Discov. Today 9 (2004) 276e285.
[60] E.J. Matthews, N.L. Kruhlak, R.D. Benz, J.F. Contreras, Curr. Drug. Discov.
Technol. 1 (2004) 61e76.
[61] L.G. Valerio, K.B. Arvidson, R.F. Chanderbhan, J.F. Contreras, Toxicol. Appl.
Pharmacol. 222 (2007) 1e16.
[62] B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan,
M. Karplus, M. Charmm, J. Comput. Chem. 4 (1983) 187e217.
[63] A. Morreale, F. Maseras, I. Iriepa, E. Gálvez, J. Mol. Graph. Modell. 21 (2002)
111e118.
[54] J.D. Irvine, L. Takahashi, K. Lockhart, J. Cheong, J.W. Tolan, H.E. Selick, J. Pharm.
Sci. 88 (1999) 28e33.
[55] S. Yee, Pharm. Res. 14 (1997) 763e766.
[64] M. Bartolini, M. Pistolozzi, V. Andrisano, J. Egea, M.G. López, I. Iriepa,
I. Moraleda, E. Gálvez, J. Marco-Contelles, A. Samadi, ChemMedChem 6 (2011)
1990e1997.
[56] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Eur.
J. Pharm. Sci. 10 (2000) 195e204.